Smad7-based biologic targeting epidermis and stroma promotes healing of diabetic wounds in mice and pigs

Domain: biology
Published: 2026-03-26
Editorial outcome: major_revision

Abstract
Diabetic wounds have limited effective therapies to restore tissue repair and resolve
excessive inflammation. This study aimed to identify mechanisms of diabetic wound
healing defects and test a therapeutic intervention using diabetic mouse and pig models.
Here we show that Smad7 transgene expression in mouse epidermis promotes wound
healing in diabetic mice. To restrict therapeutic effects of Smad7 on wounds, we develop
a Smad7-based biologic (Tat-PYC-Smad7) that penetrates cells of the wound. Topical
Tat-PYC-Smad7 treatment to diabetic pig and mouse wounds accelerates healing. Tat-
PYC-Smad7-treated wounds exhibit reduced TGFβ/NFκB signaling, faster re-
epithelialization and better extracellular matrix remodeling compared to vehicle controls.
Tat-PYC-Smad7 also attenuates neutrophil ext racellular trap (NET) formation, potentially
acting through reductions in MPO enzymatic activity and MPO nuclear entry,
consequently reducing chromatin Ldecondensation and the release of NET components.
Our study reveals that keratinocytes and neutrophils are the two major cell types targeted
by Tat-PYC-Smad7 to pRromote diabetic wound healing, providing insight into mechanisms
of diabetic wound healing defects targetable by Smad7-based therapy.

Introduction
Impaired wound healing in diabetes represents a significant cause of morbidity 1.
Standard wound management is often ineffective, posing global health, economic, and
social issues 2, 3. Becaplermin/Regranex, a recombinant platelet-derived growth factor
(PDGF)-BB, is the only FDA-approved topical drug for diabetic foot ulcers 4. However,
Regranex requires large, repetitive doses to achieve modest improvement in a small
proportion of patients 5. In addition, long-term use of growth factors is linked to a
carcinogenesis risk 6. Hence, developing novel, effective treatments for diabetic wounds
is an unmet medical need.

The wound healing process includes stages of inflammation, proliferation, and
remodeling 3. In diabetic wounds, healing stal ls at the inflammation phase 7. One of the
hallmarks of nonhealing human wounds is keratinocyte dysfunction 8, which has been
considered a therapeutic targetL for diabetic wounds9. Prolonged inflammation in
nonhealing wounds generates reactive oxygen species (ROS), leading to keratinocyte
injury, apoptosis, and imRpaired migration 10. Moreover, altered extracellular matrix (ECM)
composition and enhanced ECM degradation by elevated matrix metalloproteinase levels
in nonhealing wounds impair keratinocyte attachment, leading to aberrant cell signaling
and impaired survival and migration 11. In addition to keratinocyte dysfunction, prolonged
infiltration and activation of inflammatory cells, mainly neutrophils, are the main source of
proinflammatory cytokines, proteases, and ROS in chronic wounds 12, and contributes to
the recruitment of more immune cells, alterations in the proteolytic balance, and impaired
blood vessel formation 13. Collectively, keratinocyte dysfunction, sustained neutrophil-
driven inflammation, proteolytic imbalance, and impaired angiogenesis delay wound
healing, exacerbate scar formation, and predispose local lesions to neoplastic
progression 7, 14. In patients with diabetes, elevated glucose levels promote neutrophil
extracellular trap (NET) formation through the process of NETosis 15. The pathogenic role
of prolonged neutrophil activity in diabetic wounds potentially serves as a therapeutic
target. Conversely, individuals with neutropenia or those suffering from conditions with
neutrophil abnormalities (such as deficiencies in adhesion or neutrophil granules, etc.)
are more susceptible to wound infections resulting in abnormal wound healing processes
16. The impaired wound healing observed in neutropenia and neutrophil dysfunction
suggests that a systemic approach to targeting neutrophils may not be viable because
neutrophils play dual roles in wound healing.

To study mechanisms of healing defects in diabetic wounds and test therapeutic
interventions, animal models have been dev eloped 17. Excisional wounds in diabetic
BKS.Cg-Dock7m +/+ Leprdb/J (db/db) mice have a significant healing delay, with
reductions in neovascularization aLnd epidermal proliferation 18. Miniature diabetic pigs
are also a model for wound healing studies due to the similarities of their skin texture to
humans 19. While theR diabetic porcine model offers a clinically relevant model, e.g.
enabling an assessment of treatment effects on ECM remodeling, its limitations, such as
the extensive clinical care, long preparation time, and limited excisional wounds per pig
restrict its feasibility for mechanistic investigations. Therefore, mouse models provide a
complementary resource for mechanistic studies. We previously observed that transgenic
mice constitutively overexpressing wild-type TGFβ1 in keratinocytes (K5.TGFβ1wt) with
TGFβ/NFκB signaling activation exhibited a significant delay in full-thickness wound
healing, characterized by profound inflammation, keratinocyte apoptosis, and ECM
dysregulation 20, 21. We also found that TGFβ and NFκB are activated in human diabetic
wounds 21, suggesting that a TGFβ/NFκB dual inhibitor, Smad7, may have therapeutic
effects on diabetic wounds. Smad7 consists of an N-terminal and C-terminal domain, with
a linker region containing a PY motif 22. The functional activities of Smad7, including
blocking TGFβ/NFκB signaling, are attributed to its C-terminal domain and PY motif 23.
These domains of human Smad7 share 99% amino acid homology with mouse and pig
Smad7, enabling the use of these animal models to investigate its function while
minimizing species-mismatch-associated immunogenic responses 22. In this study, we
employed both genetic and pharmaceutical models to assess the role of Smad7 in
diabetic wound repair. We provide evidence that Smad7-based proteins promotes
diabetic wound healing via supporting epithelial cell survival, ECM modification, and
attenuating adverse neutrophil activities.

Results
K5.Smad7 transgene expression and Tat-PYC-Smad7 topical application mitigated
wound healing defects of db/db mice.
We previously developed K5.Smad7 mice, which express Smad7 in keratinocytes 24. We
bred K5.Smad7 mice with db/db mice that have obesity and type 2 diabetes 25, to
generate K5.Smad7/db/db bigenic mice. Grossly, K5.Smad7/db/db mice had sparse hair
(Fig.1a), similar to K5.Smad7 mice 26. The db/db phenotype of obesity and high blood
glucose levels were significantly lower in K5.Smad7/db/db mice than in db/db littermates
(Fig.1b-c, Supplementary Table 1). Microscopically, K5.Smad7/db/db skin showed mild
epidermal hyperplasia and hypertrophic sebaceous glands, similar to K5.Smad7 skin
(Fig.1a). To assess whether the K5.Smad7 t ransgene could attenuate wound healing
defects of db/db mice, we established excisional wounds by skin punch (6mm) on the
dorsal skin of mice and observeLd wound healing kinetics. Compared to db/db mice,
wound healing in K5.Smad7/db/db mice was significantly accelerated from day 9 to day
11 (Fig.1d-e).

Because K5.Smad7 mice expressed Smad7 transgene in other epithelial cells in addition
to keratinocytes 24, to assess whether Smad7 healing is a direct effect on wounds or a
secondary effect of attenuated diabetic phenotype or from other tissues, we generated a
Tat-PYC-Smad7 protein that contains the functional domains of human Smad7 with a cell-
penetrating Tat peptide. An optional HA tag was added to the C-terminus of Tat-PYC-
Smad7 enabling tracking of this exogenous protein (Fig.1f). Testing topical application of
Tat-PYC-Smad7 on normal mouse skin wounds indicated a relatively long stability of Tat-
PYC-Smad7 (at least 12h) (Fig.1g). Four full-thickness skin wounds were induced using
a skin punch on the dorsum of mice on day 1, followed by topical treatment every other
day and covered by a 3M film after treatment (Fig.1h). Tat-PYC-Smad7 penetrated both
epithelial and stromal cells of db/db wounds (Fig.1i). Grossly, we observed accelerated
wound healing in both K5.Smad7/db/db wounds and wounds of db/db mice treated with
Tat-PYC-Smad7 from day 9 to day 11 (Fig.1j-k). Histologically, K5.Smad7 transgene
expression or Tat-PYC-Smad7 treatment led to increased diabetic wound healing rates
(Fig.1l-m). Additionally, there was a reduction in the remaining wound length and an
increase in the length of the epidermal migrating tongue in K5.Smad7/db/db wounds and
Tat-PYC-Smad7-treated wounds, compared to vehicle-treated db/db wounds (Fig.1l, n,
o). Notably, Tat-PYC-Smad7 topical treatment exerted an effect comparable to K5.Smad7
transgene expression in promoting diabetic wound healing. We did not observe a
significant change in either blood glucose or b ody weight in mouse hosts with Tat-PYC-
Smad7-treated wounds compared to the vehicle control (Supplementary Fig.1).

We found a dose-dependent eLffect of Tat-PYC-Smad7 in accelerating wound healing
(Supplementary Fig.2). Then we used 1µg of Tat-PYC-Smad7, which showed the best
treatment efficacy, Rto compare with standard-of-care treatment, Regranex
(Supplementary Fig.3a). Tat-PYC-Smad7 significantly expedited wound closure
compared to either vehicle or Regranex treatment on day 5 and 7 (and comparable
healing to Regranex at later time points) and Tat-PYC-Smad7 had an overall higher rate
of healing (Supplementary Fig.3b-e). From day 8 to day 11, wounds treated with Tat-PYC-
Smad7 exhibited reduced size and longer lengths of epidermal migrating tongues
compared to vehicle (similar or better than Regranex-treated wounds) (Supplementary
Fig.3f-h). We also observed foam-like F4/80+ myeloid cells in Regranex-treated wounds
but not in Tat-PYC-Smad7-treated wounds (Supplementary Fig.4). These foam-like cells
with internalized lipids and a characteristic foamy cytoplasmic appearance may indicate
active inflammation and have been linked to a profibrotic phenotype 27. Regranex-treated
mice had increased numbers of circulating white blood cells including lymphocytes,
monocytes, and granulocytes compared to vehicle- or Tat-PYC-Smad7 treated mice
(Supplementary Fig.5a). In addition, Regranex, but not Tat-PYC-Smad7, upregulated
pro-inflammatory factors such as Angiopoietin 2, chemokines CXCL16 and CXCL5, LDL
receptor (LDL R) and IGFBP3 (Supplementary Fig.5b and c).

Tat-PYC-Smad7 promoted the healing of diabetic pig wounds.
To assess the effect of Tat-PYC-Smad7 on wounds closely mimicking human skin, we
used three miniature diabetic pigs and punched twelve 2cm diameter full-thickness
wounds in each pig for drug treatment (Fig.2a). Two doses of Tat-PYC-Smad7 protein (2
µg and 4 µg) or vehicle control of either hydro xypropyl cellulose (HPC) or hydroxyethyl
cellulose (HEC) were topically applied every other day and wounds were covered with a
medical dressing (Fig.2a). ImmunLohistochemistry staining for Smad7 revealed that Tat-
PYC-Smad7 was detected in the epidermis and stromal cells at the wound edge in
protein-treated woundsR, while it was absent in vehicle-treated wounds (Supplementary
Fig.6a). To assess whether topically applied Tat-PYC-Smad7 enters the bloodstream, a
Smad7-specific ELISA was developed, capable of detecting levels as low as 5 ng/mL.
Smad7 remained undetectable in systemic circulation in all treated pigs (Supplementary
Fig.6b). We evaluated whether Tat-PYC-Smad7 induced an anti-drug antibody (ADA)
response that could neutralize its therapeutic effects and found no detectable ADA against
Tat-PYC-Smad7 in treated pigs (Supplementary. Fig 6c). Wound healing kinetics among
individual pigs varied, resembling variations in human diabetic wounds 28, hence wound
healing response to treatment of each pig by gross evaluation was analyzed individually.
Grossly, the treatment effects of Tat-PYC-Smad7 became evident from day 8, 5 and 11
for pig 1, 2 and 3, respectively (Fig.2b-c). Histological analysis indicated that more than
30% of the wounds were healed in the Tat-PYC-Smad7-treated group compared to 8%
of the vehicle group (Fig.2d-e). Tat-PYC-Smad7 significantly reduced the remaining
wound length as a percentage of the initial wound length compared to the vehicle control,
indicating enhanced wound closure in diabetic pigs (Fig.2f). Moreover, Tat-PYC-Smad7
treatment resulted in longer epidermal migrating tongues in all three pigs compared to
vehicle controls (Fig.2g).

To understand the pathogenic targets of Tat-PYC-Smad7 in wound healing, we
performed immunofluorescent staining of diabetic pig and db/db mouse wound sections
and found that Tat-PYC-Smad7-treated wounds contain more Ki67+ cells and less αSMA+
area compared to vehicle-treated wounds (F ig.3a-b, Supplementary Fig.7, suggesting
increased keratinocyte proliferation and less fibrotic response. Moreover, Tat-PYC-
Smad7 restored angiogenesis in thLe stroma area under the migrated epidermis in diabetic
pig wounds and db/db mouse wounds, with more CD31-positive areas at the edge of Tat-
PYC-Smad7-treated wRounds compared to control wounds (Fig.3c-d, Supplementary
Fig.7b). Additionally, Tat-PYC-Smad7-treated wounds had more regenerative and
organized wavy elastic fibers (stained as black to blue/black striae) entwined among
collagenous fibers shown by Pentachrome staining in healed wounds of db/db mice on
day 25 and diabetic pig wounds on day 16 (Fig.3e-f), indicating that Tat-PYC-Smad7
improved ECM deposition and collagen remodeling.

To understand the molecular basis of Tat-PYC-Smad7 effects, we performed RNA-
seq on db/db mouse wound samples excised at identical locations treated with vehicle or
Tat-PYC-Smad7 on days 9 and 11. Days 9 and 11 are critical stages at which db/db
wounds start to transition towards a chronic state, compared to normal wounds 18.
Principal components analysis revealed distinct transcriptomic profiles of Tat-PYC-
Smad7-treated wounds vs vehicle-treated wounds (Fig.4a). The number of differentially
expressed genes (DEGs) was 211 on day 9 and 612 on day 11, with 72 DEGs overlapping
between the two timepoints (Fig.4b). Gene set enrichment analysis (GSEA) using the
Hallmark gene sets showed that Tat-PYC-Smad7 inhibited TGFβ and NFκB signaling,
inflammatory responses and apoptosis pathways (Fig.4c and Supplementary Fig.8),
consistent with known TGFβ/NFkB effects on wound healing in our previous reports 21, 29
as well as phenotypic changes seen in Fig.3a-b. Interestingly, enrichment analysis using
the overlapping DEGs between day 9 and day 11 on gene ontology (GO) terms identified
that neutrophil functions affected by Tat-PYC-Smad7 were among three out of top five
enriched biological processes (Fig.4d). As DEG enrichment analysis indicated a
significant impact of Tat-PYC-Smad7 on neutrophils on both day 9 and day 11, we
examined Ly6G+ neutrophils in theL wound beds and found that the number of Ly6G+ cells
was unchanged by Tat-PYC-Smad7 treatment on day 9, but significantly reduced on day
11 (Fig.4e-f), suggestinRg that Tat-PYC-Smad7 may affect neutrophil functionality and
chemotaxis. Analysis of wound tissue lysates from day 11 using an antibody array
revealed that wounds from mice treated with Tat-PYC-Smad7 had reduced
proinflammatory proteins and cytokines, including CXCL5 30, lipocalin2 31, and cytokine
IL33 32, and granule components myeloperoxidase (MPO) 33 and Pentraxin 3 34,
compared to wounds from mice treated with vehicle (Fig.4g-h). Tat-PYC-Smad7-treated
wounds also had decreased elastase production on day 11(Fig.4i), which is often
associated with neutrophil degranulation or NETosis 35. Therefore, Tat-PYC-Smad7 exerts
effects on both epidermis and stroma cells in diabetic wounds with better healing
outcomes.

Tat-PYC-Smad7 blunted neutrophil activities in diabetic wounds at molecular and
cellular levels.
In addition to the impact of Tat-PYC-Smad7 on keratinocytes and ECM regulation, GSEA
of GO terms on day 9 and day 11 showed the top 3 functional clusters associated with
neutrophil activities that were consistently attenuated by Tat-PYC-Smad7 treatment
(Fig.4j, Supplementary Fig.9a). This is consistent with the later-stage neutrophil infiltration
observed in db/db mouse wounds, as Ly6G staining showed only sparse neutrophil
presence on days 3 and 5 in comparison to abundant neutrophil infiltration on day 9 and
day 11 after wounding (Supplementary Fig. 9b). Immunohistochemistry staining of the
wound tissues showed penetration of Tat-PYC-Smad7 in both keratinocytes and Ly6G+
cells in protein-treated wounds (Supplementary Fig.10), indicating a possible direct effect
of Tat-PYC-Smad7 on neutrophil functions. Additionally, in diabetes, high glucose (HG)
levels induce neutrophils to undeLrgo NETosis 15. Neutrophils recruited to wound sites
almost immediately undergo NETosis and complete NET release in ~2.5 hours 15. To
evaluate the Tat-PYC-SRmad7 effect on NETosis in vivo, we examined a transcriptomic
profile of NETosis 36 previously demonstrated to be specifically involved in diabetic wound
healing defects 15. We found that the NETosis-related gene set was downregulated in
wounds treated with Tat-PYC-Smad7 (Fig.4k and l), with similarly strong enrichment as
other neutrophil-associated gene sets (Fig.4j and Supplementary Fig.9a). Furthermore,
in both diabetic pig and db/db mouse models, immunofluorescence staining indicated
fewer neutrophils undergoing NETosis in the Tat-PYC-Smad7-treated wound beds,
determined by co-staining of Ly6G or MPO (neutrophil markers) and citrullinated histone
3 (citH3) (NETosis marker) with DAPI (DNA marker) (Fig.5a-b). These data suggest that
Tat-PYC-Smad7 blunts neutrophil NETosis in diabetic wounds.

Tat-PYC-Smad7 prevented PMA and high glucose-induced NETosis in dHL60 cells.
To test if Tat-PYC-Smad7 directly affects NETosis, we performed an in vitro assay using
phorbol myristate acetate (PMA)-induced NETosis in dimethyl sulfoxide (DMSO)-
differentiated HL60 cells (dHL60), a neutrophil cell culture model 37. Tat-PYC-Smad7-HA
was detected in the nucleus 5min after treatment, showing highly efficient cell penetration
(Supplementary Fig.11a). We observed that dHL60 cells treated with Tat-PYC-Smad7 had
fewer cells marked with NETs 4 hours post-induction with PMA (Supplementary Fig.11b).
Live cell imaging using IncuCyte showed significant attenuation of PMA-induced NETosis
by Tat-PYC-Smad7 in a dose and time-dependent manner (Fig.5c-d). Cytotoxic Green, a
cell-impermeant DNA dye that labels extracellular DNA released during loss of membrane
integrity, colocalized with citH3-positive neutr ophils and MPO in PMA-induced dHL60
cells (Supplementary Fig. 12), confirming that the observed signal represented NETosis.
We also observed reduced elastasLe release from dHL60 cells 4 hours after PMA induction
following Tat-PYC-Smad7 treatment, an additional marker of NETosis (Fig.5e). NETosis
induced by HG, mimickiRng the blood glucose levels in diabetic wounds, was also inhibited
by Tat-PYC-Smad7, (Fig.5f-g). Furthermore, siRNA knockdown of endogenous Smad7 in
dHL60 cells significantly increased NETosis under both PMA- and HG-induced conditions
(Fig.5h-j), suggesting Smad7 suppresses NETosis. PMA-induced NETosis was not
affected by BSA, TGFβ or TGFβ inhibitor treatments (Supplementary Fig.13), suggesting
attenuation of NETosis by Tat-PYC-Smad7 was not related to its effect of antagonizing
TGFβ signaling or as a non-specific protein.

We next explored the molecular mechanism underlying the effect of Tat-PYC-Smad7
on neutrophil activity. As histone citrullination is the critical step for NETosis 38, we first
investigated whether Tat-PYC-Smad7 affects this process. PMA-induced citrullinated
histone 3 (citH3) was blocked by Tat-PYC-Smad7 treatment at all time points examined
(Fig.6a-b) and all doses of Tat-PYC-Smad7 blocked citrullination of histone 3 (Fig.6c-d).
PAD4, the key enzyme for histone citrullination 38, showed no significant change in protein
levels following Tat-PYC-Smad7 treatment (Fig.6c-d). We conducted a pulldown assay
with a Smad7 antibody recognizing Tat-PYC-Smad7 using lysates of PMA-treated dHL60
cells treated with the vehicle or Tat-PYC-Smad7. Mass spectrometry analysis showed
several proteins were enriched in Tat-PYC-Smad7 samples pulled down with anti-Smad7
antibody (Supplementary Fig.14a-b). A list of the top Tat-PYC-Smad7-binding targets
includes MPO (Supplementary Fig.14b), a protein required for NETosis via translocation
into the nucleus to promote chromatin unfolding 38, 39. Consistent with mass spectrometry
findings, MPO was detectable in anti-Smad 7 pulldown samples only from Tat-PYC-
Smad7/PMA-treated dHL60 cell lysates, supporting a potential interaction between Tat-
PYC-Smad7 and MPO (Fig.6e)L. Functional assays revealed that Tat-PYC-Smad7
treatment attenuated both peroxidation and chlorination activities of MPO under NETosis
condition (Fig.6f), whichR contribute to NETosis through reactive oxidant generation, lipid
peroxidation, and chromatin remodeling 40. To further investigate the impact of Tat-PYC-
Smad7 on MPO dynamics during NETosis, we performed immunofluorescence co-
staining with MPO, HA-tagged Tat-PYC-Smad7 and Lamin B1 (a nuclear membrane
marker). MPO enters the nucleus 1 hour post-PMA treatment in the vehicle condition, but
MPO is largely excluded from the nucleus in the Tat-PYC-Smad7-treated cells at this
timepoint (Fig. 6g). By 2.5 hours, Tat-PYC-Smad7-treated cells still showed reduced
nuclear MPO accumulation and extracellular spread of MPO compared with vehicle
control (Fig. 6g). We also observed instances where MPO entered the nucleus of treated
cells, where it colocalized with DNA (DAPI) and Tat-PYC-Smad7-HA within a continuous
Lamin B1 ring at 1 hour and 2.5 hours of PMA-induced NETosis (Fig. 6g). Consistently,
at 2.5 hours, colocalization between Tat-PYC-Smad7 with MPO appeared mainly in MPO-
positive nuclei in Tat-PYC-Smad7-treated and PMA-stimulated dHL60 cells
(Supplementary Fig.15). Colocalization analysis showed that about 20-30% of the strong
HA and MPO signals appeared in the same locations, based on thresholded Manders’
coefficients. The thresholded Pearson’s correlation coefficient (Rcoloc) was close to +1,
indicating that HA and MPO signals were strongly associated in signal-rich regions
(Supplementary Table 2).

Methods
Ethical statement
All animal experiments were conducted in accordance with institutional guidelines and
approved by the Institutional Animal Care and Use Committee (IACUC) of the University
of Colorado Denver Anschutz Medical Campus (IACUC protocol #258), the University of
California, Davis (IACUC protocol #23295), and Sinclair Research an Altasciences
Company (SRC) (IACUC protocol 020084).

K5.Smad7/db/db bigenic mice
K5.Smad7/db/db bigenic mice were generated by breeding previously established
K5.Smad7 mice 24with heterozygous B6.BKS(D)-Leprdb/J mice (JAX000697) or
heterozygous BKS.Cg-Dock7m+/+Leprdb/J m ice (JAX000642). The genotyping of the
bigenic mice was performed as previously described24, 56. No gender differences were
observed in our previous woundL study 21. The female mice were utilized once they
reached 8-10 weeks of age. Before the wound healing test, mice were weighed, and their
blood glucose levels weRre assessed.

Tat-PYC-Smad7 treatment preparation.
Tat-PYC-Smad7 was generated and formulated as previously described44. Tat-PYC-
Smad7 is a recombinant protein that contains the PY and C domains of Smad7 (203-
426aa) with a cell-permeable Tat tag 21, 57. An optional HA epitope at the C-terminus of
Tat-PYC-Smad7 enabled tracking of the protein. HPC (F42, 1% Klucel HF, Ashland)
containing PBS and 30% glycerol or HEC (1% Natrosol 250HX, Ashland) containing PBS
and 30% glycerol gel was used as the base gel. Vehicle and Tat-PYC-Smad7 gel were
formulated by carefully mixing protein buffer or protein stock with the base gel at a 1:1
ratio. All doses of the formulation were aliquoted and stored at -80°C. Prior to the start of
dosing, dose formulations were freshly thawed and gently inverted to mix.

db/db mouse wound model
8-12 weeks old, genetically diabetic, female homozygous BKS.Cg-Dock7m+/+Leprdb/J
(db/db) mice (JAX000642) were purchased from Jackson Laboratory. Mice were housed
in AAALAC-accredited vivarium facilities under a 12 h light/dark cycle with temperature
and humidity monitored and maintained in ranges appropriate for rodent well-being, with
ad libitum access to food and water. Mice were acclimatized for 1 week, and their blood
glucose levels were measured using a glucometer after 16 hours of fasting to confirm
levels above 300 mg/dL before wound establishment. All mice were randomly grouped to
control for minimal environmental variation. During the wounding procedure, mice were
anesthetized with 5% inhaled isoflurane, and th e dorsal surface of the mouse was shaved
with an electric shaver. The surgical site was disinfected with 70% ethanol. Two pairs of
dorsal full-thickness wounds were created using a 6 mm biopsy punch to excise the
epidermis, dermis, and panniculus carnosus tissues. After wounding, mice were placed
on a warm pad and monRitored (via toe pinch) every 15 minutes until fully recovered. Liquid
ibuprofen (0.2 mg/mL) was provided in drinking water continuously for 24 hours before
and after the procedure. Wounds were then treated and monitored every other day. For
treating wounds with Tat-PYC-Smad7, 10 µL of formulated vehicle or Tat-PYC-Smad7 gel
(0.25, 0.5, and 1 µg dose) was applied evenly to each wound using a pipette equipped
with filter tips. Regranex was administered using a clean cotton swab to ensure even
application to the wound beds. After approximately 5 minutes, once the formulation/gel
was fully absorbed, a 3M Tegaderm Transparent Film Dressing (Frame Style, 3M™ 7 x 6
cm, 3M Healthcare, MMM1624W) was applied to completely cover the four wound beds.
All treatments were administered to the mice every other day from day 1 to the endpoint
of the experiment on day 3, 7, 8, 9, 11, or 25. Digital photographs of wounds were
captured during treatment at a consistent distance with a calibration scale. Wound areas
were calculated using a customized Python script with semi-automatic segmentation
(https://github.com/libenzheng/WoundAreaQuantification). Mice were euthanized using
CO₂ asphyxiation followed by cervical dislocation. Wound tissues from identical locations
were collected for further histological evaluation, lysis, or RNA extraction. Blood samples
were collected in EDTA-coated tubes, and fresh anticoagulated blood samples were sent
to the University of Colorado's Comparative Pathology Shared Resource Laboratory for
CBC analysis. Plasma samples were collected by centrifuging blood at 1,500 rpm for 10
minutes at 4°C and stored at -80°C until further use.

Diabetic porcine wound model and Tat-PYC -Smad7 treatment
Diabetic porcine wound model was established by Sinclair Research Center, LLC
(currently Altasciences-Columbia Lpre-Clinical) following their approved animal protocols
(Auxvasse, MO). Yucatan mini-pigs (castrated males, 27–30 kg) were used for the
experiments. Diabetes Rwas induced using an established method involving a low-dose
Streptozotocin injection (50 mg/kg) 39 to achieve fasting blood glucose levels of 150
mg/dL or above at study enrollment. Animals were observed and examined by veterinary
staff throughout the entire study. Prior to the wounding procedure, animals received a
subcutaneous injection of buprenorphine sustained release (0.2 mg/kg) and an
intramuscular (IM) injection of ketoprofen (2.2 mg/kg) as analgesic treatment. For
prophylaxis, Excede® (5 mg/kg, IM) and an intravenous injection of cefazolin (20 mg/kg)
were administered as antibiotics. On experimental day 1, animals were pre-anesthetized
with atropine (0.04 mg/kg, IM) and a combination of tiletamine/zolazepam (Telazol) and
xylazine (2.2 mg/kg, IM). Each animal was intubated endotracheally and maintained using
5% isoflurane. Twelve full-thickness 2 cm diameter excisional wounds (6 sites per side)
were established along the dorsal column of the spine using a skin punch. Immediately
after wounding, hemostasis was achieved by applying gauze sponges soaked in
epinephrine solution (1:10,000 diluted in 0.9% sterile saline). The area around the wounds
was wiped clean to ensure the peri-wound area was dry prior to treatment. For Tat-PYC-
Smad7 treatment, each wound was covered with a 3M Tegaderm Transparent Film
Dressing (Frame Style, 3M™ 7 x 6 cm), and the dose formulation was applied by injecting
Tat-PYC-Smad7 or vehicle gel through the film. After dose application, a second layer of
Tegaderm was applied to the wounds, followed by an adhesive dressing pad. The entire
wound area was covered with a foam pad and vet wrap to secure the dressing materials.
During dressing changes, the area surroundi ng the wound was moistened with sterile
saline to facilitate dressing removal and cleansed with 70% alcohol. All wounds were
gently irrigated with sterile saline Land wiped with gauze prior to dose administration. All
pigs were dosed every other day from experimental day 1 to termination on day 16. The
treatment dose started Rat 0.5 mL per wound, adjusted to 0.25 mL per wound on day 13
as the wound size decreased, and reduced to 0.1 mL per wound on day 16. Digital
photographs of the wound sites were taken before each dose administration, with every
image including a colored ruler as a scale bar and appropriate animal identifiers (animal
ID, site number, and study day/date of collection). Wound areas were quantified using
cellSens Dimension software (Olympus, version 2.3). Animals were sacrificed two hours
after the final treatment dose on post-wounding day 16. Wound samples were collected
for histological evaluation. Serum samples were collected pre-treatment (Day 0 or Day 1)
and post-treatment (Day 16) from blood drawn without anticoagulants. After allowing the
blood to clot at room temperature for 60 minutes, the samples were centrifuged at 3,000
rpm for 10 minutes at 4°C and stored at -80°C until further use.

ELISA to detect systemic Tat-PYC-Smad7 from local application.
We generated a sandwich ELISA to detect Tat-PYC-Smad7 following a customized
protocol 44. Rabbit anti-Smad7 (Novus, NBP1-87728) was coated onto 96-well plates
(Nunc-Immuno, 446612) overnight and served to capture Smad7 from standards and
samples. Mouse monoclonal antibody (R&D, MAB2029) that recognizes the c-terminus
of human Smad7 was used for detection. We used HRP-conjugated horse anti-mouse-
IgG (Cell Signaling Technologies) to detect captured immune complexes and 1-Step Ultra
TMB-ELISA substrate (Thermo Fisher Scientific) to detect HRP activity, followed by acid
quenching and detection on a plate reader at OD450. We spiked Tat-PYC-Smad7 into
untreated pig serum to establish a standard c urve and compared Smad7 levels in pig
serum samples pre- (Day 0 or Day 1) and post- (Day 16) Tat-PYC-Smad7 treatment.

Anti-Drug Antibody (ADA) ELISAL
An ADA ELISA was developed to assess anti-Smad7 antibodies in pig serum. Mouse anti-
Smad7 antibody (R&D RSystems, MAB2029) was used to generate a reference standard.
The assay conditions were optimized by titrating secondary antibodies to minimize cross-
reactivity while generating similar on-target signal using anti-mouse HRP (Cell Signaling
Technology, 7076) at 1:5,000 and anti-pig HRP (Jackson ImmunoResearch, 114-035-003)
at 1:25,000. 96-well microplates (Immulon 4B, Thermo Fisher Scientific, 3855) were
coated with recombinant Smad7 protein (20 µg/mL in PBS) and incubated overnight at
4°C. Wells were blocked with casein-based blocking buffer (Thermo Scientific, PI37528)
for 1 hour at room temperature, followed by addition of three-fold serial serum dilutions
(1:30, 1:90, 1:270, 1:810, 1:2430, 1:7290, 1:21870 and 0). Bound antibodies were
detected using anti-mouse HRP (1:5,000) and anti-pig HRP (1:25,000), followed by TMB
substrate (Sigma, T8665) and 1M sulfuric acid stop solution. OD450 was measured using
a Bio-Rad iMark Microplate Reader. ADA positivity was determined based on OD450
values exceeding background levels and aligning with the reference standard.

Immunofluorescence (IF), Immunohistochemistry (IHC), Multispectral Imaging (MSI)
and Immunocytochemistry (ICC)
IF/IHC/MSI staining was performed on formalin-fixed paraffin-embedded (FFPE) skin
sections. ICC was conducted to detect protein expression and localization in cultured
cells. The IF and IHC staining procedures followed established protocols51. Sections were
deparaffinized, rehydrated, subjected to heat-induced antigen retrieval, and blocked prior
to incubation with primary antibodies. After primary antibody incubation, IF slides were
stained with Alexa Fluor-conjugated sec ondary antibodies (Invitrogen™) and
counterstained with DAPI. IHC slides were treated with SignalStain® Boost IHC Detection
Reagent (HRP, Rabbit) (Cell SignaLling Technology, catalog number 8114) or SignalStain®
Boost IHC Detection Reagent (HRP, Goat) (Cell Signaling Technology, catalog number
63707), followed by viRsualization using the DAB Quanto Detection System (Epredia,
catalog number TA-125-QHDX). For co-staining of Ly6G/CitH3/DAPI and
MPO/CitH3/DAPI, MSI was performed using the Opal™ 4-Color Fluorescent IHC Kit
(Akoya Biosciences, catalog number NEL810001K) according to the manufacturer's
instructions. For ICC, 1 million cells were seeded into Ibidi eight-well removable slides
precoated with 0.01% poly-L-lysine solution (Sigma-Aldrich) and allowed to adhere for 20
minutes. Cells were stained using the Immunofluorescence Application Solutions Kit (Cell
Signaling Technology, 12727) following the manufacturer's protocol. Primary antibodies
used for IF/IHC/MSI/ICC are listed in Supplementary Table 3. Antibodies were validated
for specificity using appropriate positive and negative controls. Where applicable,
specificity was confirmed using tissues or protein lysates as positive controls based on
detection of bands at the expected molecular weight by immunoblotting. Consistent
staining patterns and signal specificity were verified across independent experiments.
Secondary antibodies for IF and ICC included Alexa Fluor™ 488-conjugated anti-mouse
IgG (Invitrogen™, A-11001), Alexa Fluor™ 594-conjugated anti-rabbit IgG (Invitrogen™,
A-11012), and Alexa Fluor™ 647-conjugated anti-rabbit IgG (Invitrogen™, A-21244).
After staining, MSI/IF/ICC slides were mounted with ProLong™ Gold Antifade Mountant
(Invitrogen™, P36930) and cured in the dark at room temperature for 24 hours. The slides
were then stored at 4°C until imaging.

Imaging and Quantification
Imaging was performed using an Olympus IX83 microscope system for IF/ IHC/MSI slides,
while confocal imaging was conducted using the Leica TCS SP8 STED 3X. For image
quantification, sequential 10x orL 20x images were captured along the basement
membrane, with three images per slide. Quantification was performed using cellSens
Dimension software (ORlympus, version 2.3), and the average of the three images was
considered the representative value for each sample slide. Nuclear Ki67-positive cells
were quantified as the percentage of positive cell area relative to the epidermal area
delineated by keratin 14 staining. CD31-, Ly6G/CitH3/DAPI-, and MPO/CitH3/DAPI-
positive cells were quantified as the percentage of positive cell area relative to the total
skin area within the wound area. The quantification of F4/80⁺ foam-like cells was
performed using Count&Measure function of cellSens Dimension software (Olympus,
version 2.3) based on hue, saturation, and value (HSV) thresholding and morphological
criteria. To specifically identify foam-like F4/80⁺ cells, images were processed using an
HSV segmentation mask with the following threshold parameters: Hue: 4–36, Saturation:
35–101, Value: 158–218. The minimum object size was set to 15 pixels. Colocalization
analysis was performed using the Colocalization Threshold plugin in Fiji (ImageJ) on
confocal images of HA and MPO. Automatic thresholding (Costes method) was applied
to remove background noise. Thresholded Pearson’s correlation coefficient (Rcoloc;
range: –1 to +1) measured linear correlation within signal-positive regions above
threshold, with values near +1 indicating strong colocalization. Thresholded Manders’
coefficients (M1 and M2) quantified the percentage of HA or MPO signal overlapping with
the other above threshold, ranging from 0% (no overlap) to 100% (complete overlap),
providing a refined assessment of specific, biologically relevant colocalization.

Trichrome Staining and Movat Pentachrome Staining
Trichrome staining and Movat Pentachrome staining were performed using Trichrome
Stain Kit (Abcam, ab150686) and Movat Pentachrome Stain Kit (Abcam, ab245884)
following the manufacturer’s insLtructions. Elastic fibers were identified as black to
blue/black striae in Movat Pentachrome staining.

RNA-Seq Data AnalysRis
Tissue samples for RNA-Seq analysis were collected, rapidly frozen in liquid nitrogen,
and stored at -80°C. RNA extraction was performed using an RNA Plus mini-Kit from
Qiagen (Qiagen, 74136). The RNA sequencing and library preparation was conducted at
the University of Colorado Denver Cancer Center's Genomics Shared Resource.
NuQuant Universal Plus mRNA seq kit was used to prepare mRNA sequencing libraries.
Sequencing was performed on an Illumina NovaSEQ 6000 with 2x150 paired-end reads
at 80 million reads per sample depth. Quality control was conducted by FastQC
(https://www.bioinformatics.babraham.ac.uk/projects/fastqc/) and FastQ Screen
(https://www.bioinformatics.babraham.ac.uk/projects/fastq_screen/). The RNA-seq reads
were processed with BBDuk (BBMap – Bushnell B. – sourceforge.net/projects/bbmap/)
and aligned to mouse genome GRCm38.p6 (release 96) with STAR 58. The differential
expression analysis was performed on TMM (trimmed-mean M values) normalized count
data using negative binomial generalized linear model and likelihood ratio test59,60
implemented in the edgeR package (version 4.0.11,
https://doi.org/10.5281/zenodo.3748085)61. The Benjamini–Hochberg multiplicity
correction was applied on p-values to calculate false discovery rates. GSEA was
performed using pre-ranked GSEA62 and Enrichr 63 implemented by the GSEApy
package (version 1.1.2, https://doi.org/10.5281/zenodo.3748085), with pathways from
GO biological process, KEGG, and hallmark gene set collection from the Molecular
Signatures Database. The pre-ranked GSEA utilized all identified genes pre-ranked by
the negative log transformed p-values with the sign of log2 fold change. The Enrichr
handled a list of differentially expreLssed genes with p-values less than 0.05 and absolute
log2 fold changes greater than 0.6

Proteome profiler antiRbody arrays
Tissue lysates from db/db mouse wounds and plasma samples were analyzed using
Proteome Profiler Antibody Arrays (R&D Systems). For tissue lysis, wound tissues were
placed in RIPA buffer (Cell Signaling Technology, 9806) supplemented with Protease
Inhibitor Cocktail (Roche, 4693124001). The tissues were finely cut, homogenized, and
centrifuged at 10,000g for 10 minutes at 4°C to remove debris. Protein concentrations
were measured using the Pierce™ BCA Protein Assay Kit (ThermoFisher, 23225) and
adjusted to equal amounts before analysis. Plasma samples were collected from
euthanized mice at the endpoint of the wound study as described above. Tissue lysates
(500 µg) or freshly thawed mouse plasma samples (200 µL) were analyzed using the
Mouse XL Cytokine Array Kit (R&D Systems, ARY028). After blocking, samples were
incubated with spotted nitrocellulose membranes according to the manufacturer’s
instructions. Membrane HRP luminescence intensities were visualized using a ChemiDoc
Imaging System (Bio-Rad), with an exposure time of 5 min, and images were saved as
high-resolution TIFF files. The registered intensity of each dot was determined in
duplicates by Quick Spots software (Ideal Eyes Systems), with background signals from
blank control spots subtracted. Four biological replicates were measured and analyzed
per condition.

Cell culture, treatment and siRNA transfection
HL60 cells purchased from the University of Colorado Cancer Center Cell Technologies
Resource were maintained in RPMI 1640 med ium (Gibco) supplemented with 10% FBS
and 1% Primocin™ (InvivoGen). Cells were differentiated with culture media
supplemented with 1.3% DMSO (SLigma, D2650) as previously described37. Differentiated
HL60 cells (dHL60) were used 3 or 7 days after the initiation of differentiation. For PMA-
induced NETosis, dHL60 were incubated with size exclusion chromatography (SEC)
buffer (250 mM NaCl, 25 mM NaPO , pH 7.6) as vehicle control, or with Tat-PYC-Smad7
4
or BSA at the dose of 3, 5, and 10µg/mL (in SEC buffer) for 10min, or with recombinant
human TGFβ1 (R&D, 7754-BH) at the dose of 2, 5ng/mL or TGFβ inhibitor LY2109761 at
the dose of 1µg/ml for 48h, before adding 20nM or 100nM PMA to induce NETosis. For
high-glucose experiments, differentiated HL60 (dHL60) cells were incubated in Ham’s F-
12K (Kaighn’s) Medium culture medium (Gibco, 21127022) containing either normal
glucose (NG) (7 mM, 1.260 g/L) or high glucose (HG) (33 mM) for 6 hours. A glucose
concentration of 33 mM (equivalent to 594 mg/dL) is similar to the fed blood glucose level
in BKS.Cg-Dock7m+/+Leprdb/J (db/db) mice. Mannitol (26 mM in medium with NG) was
used as an osmotic control. Cells were then treated with Tat-PYC-Smad7 at
concentrations of 3 and 5 µg/mL or vehicle control (SEC buffer).

For siRNA transfection, dHL60 cells were seeded at a density of 300,000 cells per
well in a 6-well plate after three days of differentiation. A total of 0.02 nmol of ON-
TARGETplus Human Smad7 siRNA-SMARTpool (L-020068-00-0005, Horizon
Discovery/Dharmacon) or 0.02 nmol nontargeting siRNA pool (D-001810-10-05, Horizon
Discovery/Dharmacon) was transfected using 1.2 µL of Lipofectamine™ RNAiMAX
Transfection Reagent (Thermo Fisher, 13778100) per well. A subset of cells was
harvested 48 hours post-transfection for RNA extraction and RT-PCR, as well as for cell
lysis and western blotting to evaluate knockdown efficiency. The remaining cells were
treated with HG or PMA to induce NETosis an d monitored using IncuCyte, as described
below.

Imaging and quantification of NETosis using SIEVEWELL
dHL60 cells were incubated for 5 minutes with the membrane-permeable NUCLEAR-ID
Red DNA dye (Enzo LifRe Sciences, ENZ52406) to stain nuclei. Following three washes,
NUCLEAR-ID Red-stained dHL60 cells were loaded into SIEVEWELL chambers (TOK,
Japan). To induce and assess NETosis, RPMI medium containing 200 nM Cytotox Green
Reagent (Sartorius, 4633) and 100 nM PMA (Cayman Chemical, 10008014) was added.
After 3 hours, SIEVEWELL slides were imaged using an Olympus IX83 microscope.
Sequential 20x images per slide were quantified using cellSens Dimension software
(Olympus, version 2.3). Co-localization of red and green signals was considered as
objects undergoing NETosis.

Live-cell NETosis observation using the IncuCyte
For the NETosis assay, the ImageLock plate was coated with 0.1μg/mL fibronectin (Sigma,
F1141) for 1 hour. dHL60 (100μL per well, 200, 000 cells) was incubated in 250nM Cytotox
Green Reagent in a low fluorescence media Ham’s F-12K (Kaighn’s) Medium (Gibco,
21127022). Cells were scanned at the baseline level (0h). Live-cell imaging was taken
every 10 or 30 minutes or 1 hour by IncuCyte® Live-Cell Analysis System (Sartorius) for
at least 6 hours. The data was processed using IncuCyte® software (Sartorius).

Cell pellet collection and embedding
dHL60 cells were incubated with either SEC control or Tat-PYC-Smad7 (5 µg/mL) for 10
minutes, followed by treatment with 100 nM PMA to induce NETosis for 1 hour or 2.5
hours. Cell suspensions were collected into 1.5 mL microcentrifuge tubes and centrifuged
at 1,500 rpm, 18°C for 5 minutes. The resulting cell pellets were fixed with 10% neutral
buffered formalin (NBF) for 15 minutes, washed twice with 70% ethanol, and resuspended
in fresh 70% ethanol. Pellets were then embedded in Histogel, processed into paraffin
blocks, and sectioned for histologiLcal analysis.

Elastase Detection
The concentration of elastase in cell supernatants or tissue lysates was measured using
the NETosis Assay Kit (Abcam, ab235979) to detect the elastase production. dHL60 cells
were incubated with either SEC buffer or Tat-Smad7 protein (10 µg/mL) for 10 minutes
before being treated with PMA (100 nM) for 4 hours to induced NETosis. After washing,
the cells were treated with S7 Nuclease for 15 minutes. Supernatants from each well were
collected and assayed for neutrophil elastase. Prepared cell supernatants or tissue
extracts from wounds (1 µg/mL) were incubated for 2 hours at 37°C in 200 µL of 0.1 M
HEPES buffer (pH 7.5) containing 0.5 M NaCl, 10% DMSO, and 0.1 mM elastase
substrate. A standard curve for substrate degradation was generated using neutrophil
elastase, ranging from 0 to 36 mU per well. Substrate degradation was quantified by
measuring absorbance at 405 nm.

Quantitative Real-Time PCR
Total RNA was extracted from cells or tissue samples using the RNeasy Plus Mini Kit
(Qiagen, Cat. No. 7413R4). cDNA was synthesized from 0.02–2 µg of total RNA using the
High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, 4368814). Relative
gene expression levels were determined using TaqMan™ Gene Expression Assay (FAM)
(Thermo Fisher Scientific, 4331182) with TaqMan™ Fast Advanced Master Mix (Applied
Biosystems, 4444557), and reactions were performed on a QuantStudio 5 Real-Time
PCR System. The TaqMan Gene Expression Assays used in this study included Human
SMAD7 (Assay ID: Hs00998193_m1) and Human GAPDH (Assay ID: Hs02758991_g1).
The comparative Ct (ΔΔCt) method was applied to calculate relative fold changes in gene
expression, with GAPDH serving as the endogenous control.

Western Blotting
Protein samples were harvested with RIPA buffer (Cell Signaling Technology, 9806)
supplemented with a protease inhibitor cocktail (Roche, 4693124001). Western blotting
was performed using standard protocols 64 and detected using a ChemiDoc Imaging
System (Bio-Rad). Antibodies used in western blotting are listed in Supplementary Table
3. The grayscale intensity of protein bands was quantified using ImageJ software.
Relative expression levels were determined by normalizing the intensity of each target
protein band to its corresponding loading control band. These normalized values were
then adjusted relative to the highest intensity band, which was set to 1.

Immunoprecipitation
dHL60 cells were incubated with 10 µg/mL Tat -PYC-Smad7 protein or SEC buffer for 10
minutes before adding PMA (20 nM) to induce NETosis. Cells were lysed in cell lysis
buffer (Cell Signaling Technology, 9803) supplemented with freshly added protease
inhibitors (Roche, 4693124001). For immunoprecipitation, 1 µg of anti-Smad7 antibody
(recognizing C-terminaRl Smad7, Novus, NBP1-87728) or 1 µg of rabbit polyclonal IgG
(Novus, NB810-56910) was added to 500 µg of whole-cell lysate. Immunoprecipitation
was performed by incubating the mixture with 20 µL of prewashed protein A magnetic
beads (Cell Signaling Technology, 73778), followed by five washes with 500 µL of 1X cell
lysis buffer (Cell Signaling Technology, 9803). The pellet was resuspended in 20-40 µL of
3X SDS sample buffer, vortexed briefly, and centrifuged. The sample was then heated to
95-100°C for 5 minutes. Beads were separated using a magnetic rack, and the
supernatant was transferred to a new tube. An aliquot of 15–35 µL was loaded onto an
SDS-PAGE gel for protein separation, followed by transfer to a nitrocellulose membrane
for western blot analysis. Immunoprecipitated samples were also submitted to the Mass
Spectrometry Proteomics Shared Resource Facility for proteomic analysis.

Mass Spectrometry Proteomics
Sample preparation
Protein samples were separated using NuPAGE Bis–Tris 4–12% gradient gels (Invitrogen)
with BenchMark™ Protein Ladder (Invitrogen) as a molecular weight standard.
Electrophoresis was performed in MES SDS running buffer using an X-Cell II Mini Gel
system (Invitrogen) at 200 V, 120 mA, and 25 W per gel for 30 min. Gels were stained
with SimplyBlue™ SafeStain (Invitrogen) and subsequently destained in water according
to the manufacturer’s instructions.

Each lane was excised into three equal sections, and proteins were subjected to in-
gel digestion. Gel pieces were first destained in 25 mM ammonium bicarbonate containing
50% (v/v) acetonitrile, followed by washing with 50% acetonitrile. Disulfide bonds in
proteins were reduced with 10 mML dithiothreitol at 60 °C for 30 min and alkylated with 20
mM iodoacetamide at room temperature in the dark for 45 min. Gel fragments were then
rinsed with distilled waRter, dehydrated with acetonitrile, and dried using a SpeedVac
concentrator (Thermo Fisher Scientific). Trypsin (100 ng per sample) was added to
rehydrate the gel pieces at 4 °C for 45 min, followed by overnight digestion at 37 °C.
Peptides were acidified with formic acid to a final concentration of 1% and extracted twice
using 1% formic acid in 50% acetonitrile. Combined extracts were concentrated by
vacuum centrifugation and desalted using Pierce C18 tips (Thermo Scientific).

Mass spectrometry analysis
Peptide samples (20 μL) were loaded onto Evotips (Evosep) for desalting, rinsed with 0.1%
formic acid, and maintained in 0.1% formic acid prior to analysis. Peptide separation was
performed on an Evosep One system (Evosep, Odense, Denmark) using a PepSep
analytical column (150 μm inner diameter, 15 cm length) packed with ReproSil C18 resin
(1.9 μm, 120 Å).

The liquid chromatography system was coupled to a timsTOF Pro mass spectrometer
(Bruker Daltonics) via a CaptiveSpray nano-electrospray ion source. Data were acquired
in parallel accumulation–serial fragmentation (PASEF) mode. Ion mobility separation
ranged from 0.7 to 1.50 Vs/cm², and MS/MS spectra were collected over an m/z range of
100–1700. Each acquisition cycle included 10 PASEF MS/MS scans with a ramp time of
100 ms. Precursor ions were isolated within ±1 Th and fragmented using a collision
energy that increased linearly from 20 to 59 eV. Ions exceeding an intensity threshold of
500 counts were selected for fragmentation, with dynamic exclusion applied for 0.4 min
after reaching a target intensity.

Database Searching and Protein Identification
Raw data files were processed using MSConvert (ProteoWizard, version 3.0) to
generate .mgf files. Spectral matching was performed using Mascot (version 2.5) against
the UniProt human proRtein database. Search parameters included a precursor mass
tolerance of ±15 ppm and a fragment ion tolerance of ±0.04 Da. Trypsin was specified as
the digestion enzyme, allowing up to one missed cleavage. Variable modifications
included methionine oxidation, N-terminal acetylation, and pyroglutamate formation, while
carbamidomethylation of cysteine residues was set as a fixed modification.

Peptide and protein identifications were validated using Scaffold (version 4.9;
Proteome Software). Peptides were accepted at a probability threshold of greater than
95% based on the PeptideProphet algorithm, and proteins were required to meet a
probability threshold of greater than 99% with at least two unique peptides.

MPO activity assay
The EnzChek MPO Activity Assay Kit (E33856, Invitrogen) was used for the rapid and
sensitive determination of MPO chlorination activity and peroxidation activity using cell
lysates from dHL60 cells treated with Tat-PYC-Smad7 or Vehicle control during PMA-
induced NETosis. The fluorescence intensity of each sample was measured with a
fluorescence microplate reader using excitation at 485 nm and emission at 530 nm for
chlorination activity, and excitation at 530 nm and emission at 590 nm for peroxidation
activity. The background fluorescence measured for each zero-MPO control reaction was
subtracted from each fluorescence measurement before plotting.

Statistics & reproducibility
Statistical analyses were performed using Gra phPad Prism 10. Outliers were identified
using the ROUT method (Q = 1%), and data normality was assessed using the Shapiro-
Wilk test. For normally distributed datasets, two-tailed unpaired t tests were used for
pairwise comparisons between two groups. For non-normally distributed datasets, the
Mann-Whitney test wasR applied.

For experiments involving multiple groups or time-course analyses, data were
analyzed using one-way ANOVA or two-way repeated-measures ANOVA, followed by
Tukey’s or Šídák multiple-comparison tests, as specified in the figure legends.
Contingency data were analyzed using Fisher’s exact test. Sample sizes were determined
based on prior studies using similar experimental models21. No statistical method was
used to predetermine sample size. No data were excluded from the analyses except for
outliers identified by the criteria described above. Diabetic pig experiments were
randomized, and investigators were blinded to group allocation during experiments and
outcome assessment. Mouse gross wound measurements were quantified using
unsupervised analysis. All experiments were independently repeated at least two to three
times with similar results unless otherwise stated. For in vivo studies, each experimental
group typically consisted of 4 – 15 animals, depending on experimental design. For in
vitro experiments, each condition included at least three biological replicates per
experiment. Representative images and quantitative analyses reflect reproducible
findings across independent experiments. In figures we use the following symbolic
representation of ns, not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ***P < 0.0001. In
the figure legends we include the precise P value for all statistical tests where P ≥ 0.0001
with four digits after decimal. Exact P values are reported in the figure legends or in the
corresponding Source Data files when multiple comparisons were performed. Data are
presented as mean ± SD or mean ± SEM, as i ndicated in individual figure legends.

Data Availability
RNA-seq data generated in this sLtudy are available in the Gene Expression Omnibus
(under accession code GSE274513)
[https://www.ncbi.nlm.nRih.gov/geo/query/acc.cgi?acc=GSE274513]. Mass spectrometry
data generated in this study are available in MassIVE under accession code
(MSV000095545)[https://massive.ucsd.edu/ProteoSAFe/dataset.jsp?task=d2212a53e3
6d40ad9b872f22d98814b4]. Source Data are provided with this paper.

Code Availability Statement
A customized Python script for semi-automatic wound area segmentation has been
deposited and is publicly accessible at https://zenodo.org/records/18341887. For
RNAseq analysis, Quality control was conducted by FastQC
(https://www.bioinformatics.babraham.ac.uk/projects/fastqc/) and FastQ Screen
(https://www.bioinformatics.babraham.ac.uk/projects/fastq_screen/). The RNA-seq reads
were processed with BBDuk (BBMap – Bushnell B. – sourceforge.net/projects/bbmap/)
and aligned to mouse genome GRCm38.p6 (release 96) with STAR
[https://www.ensembl.org/Mus_musculus/Info/Index] 58. The differential expression
analysis was performed on TMM (trimmed-mean M values) normalized count data using
negative binomial generalized linear model and likelihood ratio test 59,60 implemented in
the edgeR package (version 4.0.11, https://doi.org/10.5281/zenodo.3748085) 61. The
Benjamini–Hochberg multiplicity correction was applied on p-values to calculate false
discovery rates. GSEA was performed using pre-ranked GSEA 62 and Enrichr 63
implemented by the GSEApy package (version 1.1.2,
https://doi.org/10.5281/zenodo.3748085), incorporating pathway collections from the
Molecular Signatures Database (MSigDB) [ht tps://www.gsea-msigdb.org/gsea/msigdb],
including Hallmark, KEGG, and Gene Ontology biological process gene sets.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This study makes a detailed animal experiment to verify the application value of Tat-PYC-Smad7 in diabetic wounds, and the research conclusions are worth appreciating. However, there are still some problems that need to be further improved. 1. There are duplications in references 21 and 22. 2. A previous study has reported the feasibility of using exogenous Smad7 below an oncogenic level to alleviate skin inflammation and wound healing defects associated with excessive activation of TGF-β and NF-κB. In my view, the ideas of this study coincide to some extent with those of this previous study. In addition, Tat-PYC-Smad7 reduced neutrophil infiltration and attenuated TGF-β and NFκB signaling pathways were also reported (PMID: 34610386). It is necessary to add and highlight the discussion about the innovation points of this study. 3. The border of the picture is cropped, as in Fig 1N, please check 4. Whether data on blood glucose, body weight, etc. of mice treated with topical application of Tat-PYC-Smad7 were recorded, to better illustrate whether smad7 has a direct or indirect effect in regulating healing. 5. Quantitative analysis and description of foam-like F4/80+ myeloid cells are required in Sup Fig 2. 6. How was the dose of Tat-PYC-Smad7 protein determined in animal experiments 7. On line 191, fig5E-F is incorrectly labeled. Authors should check their articles more carefully. 8. Please describe the details in the data availability", "reviewer_2": "The manuscript \"Smad7-based biologic targeting epidermis and stroma promotes healing of diabetic wounds in mice and pigs' reports on therapeutic effects of recombinant topical Sad7 in various animal models. The therapeutic effects of Smad 7 on oral epithelium in animal models has been shown before. In the current manuscript the authors use diabetic mouse and porcine models to evaluate effects of Tat-PYC-Smad7 on cutaneous wounds. There are multiple issues with the study design, and the data shown in the current manuscript that need to be clarified and carefully addressed. -K5SMAD7 model indicates overexpression in other stratified epithelia, not only skin. this may contribute to observed differences in the weight, instead authors claim and overemphasize skin specific expression. -the use of adequate controls is questionable throughout the manuscript: vehicle does not represent ideal control, scramble protein would be more appropriate. Also, WT mouse should be included as a control for K5SMAD7. -the authors should comment on the use of animal models to evaluate effect of human recombinant protein. -in Figure 1A: include K5.SMAD7 mouse in F1A along with K5.SMAD7/db/db; Figure 1N: is missing x axis label. -please specify the age of mice used, K5SMAD7 controls shown are in telogen while db/db mice are in anagen phase. This is of significant concern as the healing outcomes are influenced. -TAT-SMAD7 has less effect on peripheral immune response compared to Regranex used as a control, but further characterization of systemic effect, such as circulating cytokine levels, would need to be demonstrated to claim that TAT-SMAD7 has only local effects. This and multiple other overstatements should be revised. -Figure 2D: show corresponding D1 and D16, as comparison across conditions at a single time point other than day 1 is less meaningful. In Figure 2E show % of wound closure for each condition, normalized to initial wound size. -Figure 3: more Ki67 indicates more proliferating cells – co-staining is required to conclude the effect on keratinocytes. Additional staining of fibroblasts should be included, at minimum. -the reproducibility is also an issue; mouse experiments have been replicated twice only. Porcine model requires more wounds. -details such as type of dressing used for porcine wounds are not included; what type of 3M film was used for murine wounds? -Inhibition of NETosis is novel, however it lacks mechanistic insights. Figure S1: evidence of dermabond is visualy noticable, but selectively on db/db wounds. This confounds wound healing. Figure S2: Requires detailed labeling to indicate what morphology is being referred to as foam-like, as all three conditions show F4/80+ cells. Figure S5: The authors state that ECM remodeling is reflected in CD31 staining for endothelial cells, this is not correct.", "reviewer_3": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_4": "This study proposes a SMAD7-based biological agent (TAT-PYC-SMAD7) for the treatment of diabetic wounds, which innovatively promotes keratinocyte survival and re-epithelialization by inhibiting the TGFβ/NFκB signaling pathway. The efficacy of TAT-PYC-SMAD7 has been validated not only in mouse models but also in mini-pig diabetic models that closely resemble human skin, thereby enhancing the credibility and broad applicability of the study results. Various technical approaches, including RNA-seq, proteomics, and immunohistochemistry, were employed to explore the mechanisms of action of TAT-PYC-SMAD7 at both cellular and molecular levels, revealing its role in inhibiting neutrophil activity and NETosis. Poor healing of diabetic wounds is a global health issue, and existing treatments have limited effectiveness. The SMAD7-based therapeutic approach proposed in this study demonstrates significant efficacy and has potential clinical application value. The study's design is rigorous, utilizing multiple animal models and experimental techniques, with detailed data and reliable results that effectively support the research conclusions. The research integrates knowledge from multiple disciplines such as pathology, bioengineering, and molecular biology, showcasing the advantages of interdisciplinary studies and attracting broad academic attention. Although good efficacy has been shown in animal models, the safety, efficacy, and feasibility of TAT-PYC-SMAD7 in actual clinical applications still require further validation, as there may be challenges in translation. While the study reveals the role of TAT-PYC-SMAD7 in inhibiting neutrophil activity and NETosis, the exploration of its specific molecular mechanisms remains insufficient and may require more experimental data to support. Despite the use of various animal models, the relatively limited sample size may affect the statistical significance and generalizability of the results, necessitating further expansion of the sample size for validation.", "reviewer_5": "In this manuscript, Ke et al investigate the impact of Smad7 on diabetic wound healing. They developed the K5.Smad7/db/db diabetic mouse model where keratinocytes express Smad7 and showed that these mice have enhanced wound healing rate. To evaluate the local effect of Smad7, they developed a cell permeant Smad7-based biologics -Tat-PYC-Smad7- and showed that application of this biologics enhances wound healing rate in diabetic mice and pigs. Mechanistically, the authors found that application of Tat-PYC-Smad7 on diabetic wounds leads to increased cell proliferation markers, evidence of restored angiogenesis and increased ECM deposition as well as decreased pro-inflammatory proteins and cytokines. By transcriptomic analysis, the authors showed that application of Tat-PYC-Smad7 on diabetic wounds alters the expression of genes related to neutrophils functions including migration, degranulation and NETosis. Consistently, the authors found decreased citrullinated H3 in diabetic wounds after Tat-PYC-Smad7 application. Through in vitro studies, the authors show that Tat-PYC-Smad7 enters HL60-derived neutrophils where it prevents H3 citrullination, binds to MPO and prevent its enzymatic activity and impairs NETosis. This is an exciting manuscript that nicely goes from molecular engineering to in cellulo and animal models to identify and engineer novel biologics for enhanced diabetic wound healing. • The data on TAT-PYC-Smad7 inhibiting PMA-induced NETosis is exciting. To connect this observation back to diabetes, it would be important for the authors to validate the impact of Smad7 on NETosis in more physiologically relevant situations such as when NETosis is induced with high glucose. It would also be important to show that expressing Smad7 in dHL60 cells prevents NETosis to rule out a potential side effect of the TAT-PYC-Smad7 biologics (e.g. smaller size thus higher delivery in the nucleus than endogenous Smad7). • Because NETs are described as networks of DNA with nuclear/cytotoxic proteins, the authors should demonstrate that cytotoxic green labels NETs by showing that it colocalizes with citH3 neutrophil elastase or MPO. The authors could immunostaining for these markers at the end of the live imaging period. It would also be important to show Zoomed images of figure 6 D – at this resolution, readers cannot evaluate whether the cytotoxic green signal is extracellular (e.g. occupies a larger area than the typical cell area). • Further, the authors propose that Smad7 directly binds to MPO in the nucleus thus preventing its function in NETosis. Although MPO was shown to be important for NETosis, it is still not clear how it mediates the process – for instance, MPO enzymatic activities are not required for NETosis (https://doi.org/10.1083/jcb.201006052). Was the IP in figure 6H done with the nuclear fraction to show that MPO-Smad7 interaction occurs in the nucleus? Did the author exclude the possibility that TAT-PYC-Smad could enter neutrophil granules and prevent MPO from accessing the nucleus? Immunostaining and confocal imaging could be used to assess MPO nuclear vs granules localization after PMA stimulation of TAT-PYC-Smad7 treated dHL60 cells. • The authors show that TAT-PYC-Smad7 prevents citrullination of H3 in PMA treated dHL60 cells without impacting the expression level of PAD4. This is an interesting dataset that suggests that Smad7 is important for PAD4 enzymatic activity. First, for reproducibility purposes, the authors should show and quantify repeats of the western blots shown in figure 6 F and G. In vitro experiments testing the impact of smad7 on PAD4 citrullination activity would be important to clarify how Smad7 prevents H3 citrullination and NETosis, potentially independently of Smad7-MPO interaction. • Finally, the author proposes that Tat-PYC-Smad7 prevents neutrophils degranulation. Could the author please indicate which data supports this claim? The decreased release of neutrophil elastase could also be due to the defects in NETosis since cells release neutrophil elastase during NETosis. • This reviewer appreciates the difficulties of experimenting with animals. However, the sample size of the pig dataset is very low. And some of the analysis is done with only Pig 1 in the main figure (Pig 2 and 3 are pulled in Sup fig.9 which shows a broad distribution) where the authors perform statistical test (t-test) on samples with 4 data points. This low sample size makes the t-test very unreliable. For example, in “figure 3, D)- TAT-PYC-Smad7, 4ug” one data point is much higher than the 3 other data points which pulls the mean % CD31+ area up – in such a small sample size, an outlier has a strong impact. Another example of how the small sample size impacts the conclusion of the study is in figure 5 D where the distribution of the %Ly6G+CitH3+ cells/area shows two populations which alters the mean. This data set cannot be tested with a t-test which assumes a gaussian distribution of the sample. Thus, the in vivo data (figure 3-C, B, D Fig 5 D and E) requires more data points for the statistical tests used here and the conclusions of this study to be meaningful. The authors should also show the standard deviation of their distribution instead of the standard error of the mean (SEM), which does not allow readers to appreciate the heterogeneity of the population. • A big picture question that the authors might want to discuss is the potential side effects of Tat-PYC-Smad7 biologics. If this biologic prevents neutrophil functions, and MPO activity - which is important for host defense against microbes, will the application of Tat-PYC-Smad7 enhance pathogen propagation at the wound site?"}, "author_response": "Point-by-Point Response to Reviewers’ Comments\nWe sincerely appreciate that the editorial board has given us an opportunity to revise and\nresubmit our manuscript. We are grateful for the thoughtful and constructive comments\nfrom the reviewers. We have added substantial amounts of data and revised our\nmanuscript based on reviewers’ comments and new data. Responses to reviewer’s\ncomments are provided below explaining how the concerns are addressed experimentally\nand/or conceptually. In the revised manuscript, contents directly addressing reviewers’\nconcerns are highlighted in yellow. We hope that our responses have addressed all the\nreviewers’ concerns, and the revised manuscript is strengthened significantly.\nCritiques – Responses\nReviewer #1\n1. There are duplications in references 21 and 22.\nResponse: We apologize for the error and have corrected it in the resubmission.\n2. A previous study has reported the feasibility of using exogenous Smad7 below an\noncogenic level to alleviate skin inflammation and wound healing defects associated with\nexcessive activation of TGF-β and NF-κB. In my view, the ideas of this study coincide to\nsome extent with those of this previous study. In addition, Tat-PYC-Smad7 reduced\nneutrophil infiltration and attenuated TGF-β and NFκB signaling pathways were also\nreported (PMID: 34610386). It is necessary to add and highlight the discussion about the\ninnovation points of this study.\nResponse: We agree with the reviewer’s comment that we should more clearly delineate\nconceptual innovations and novel findings of the current study compared to our previously\npublished studies. In the introduction of the revised manuscript, we have clarified the\nrationale of the current study: although our previous studies show the correlation between\nexcessive TGF-β and NF-κB signaling in human diabetic wounds and that Smad7\nameliorates those activities in experimentally TGF-β overexpressed wounds (Li et al.,\n2019), without directly testing Smad7 effects on diabetic wound milieu, we could not\nconfirm if elevated TGF-β and NF-κB play a causal role in delayed diabetic wound healing\nor are just bystanders. In the revised discussion, we have further clarified that we\nconfirmed Smad7-based therapeutic effect in diabetic wounds using two animal models\nand that TGF-β and NF-κB are among the major therapeutic targets of Smad7. We also\nidentified that the effect of Smad7 on neutrophil activity, specifically on NETosis, is\nindependent of TGF-β signaling. This effect is difficult to capture in other experimental\nmodel systems in which neutrophil infiltrations are often transient.\n3. The border of the picture is cropped, as in Fig 1N, please check.\nResponse: This error has been corrected.\n4. Whether data on blood glucose, body weight, etc. of mice treated with topical\napplication of Tat-PYC-Smad7 were recorded, to better illustrate whether Smad7 has a\ndirect or indirect effect in regulating healing.\nResponse: We recorded body weight and blood glucose levels data before the start of\nthe wound establishment and during sample collection. We found that the body weight\nand glucose levels were not significantly affected by topical Tat-PYC-Smad7 treatment.\nThese data have been included in the revised manuscript (Sup Fig. 1). We also added\nnew data showing that effects of Tat-PYC-Smad7 on diabetic pig wounds were restricted\nto wounds without systemic detectable Tat-PYC-Smad7 (Sup Fig. 6). Taken together, the\nimpact on wound healing from topical treatment is primarily through local effects. We have\nadded the related Discussion.\n5. Quantitative analysis and description of foam-like F4/80+ myeloid cells are required\nin Sup Fig 2.\nResponse: We have performed a quantitative analysis of foam-like F4/80⁺ myeloid cells\nand included the results in Sup Fig. 4B and 4C. A description of foam-like F4/80⁺ myeloid\ncells is provided in the Results section. Additionally, we have included a detailed\ndescription of the quantification protocol in the revised figure legend of Sup Fig 4 and\nSupplementary Methods.\n6. How was the dose of Tat-PYC-Smad7 protein determined in animal experiments?\nResponse: The dose of Tat-PYC-Smad7 protein was determined based on the dose-\nresponse experiments we performed. The results showed that 1µg Tat-PYC-Smad7 had\nthe best treatment effect on promoting db/db wound healing. We have added these data\nin the Sup Fig.2.\n7. On line 191, fig5E-F is incorrectly labeled. Authors should check their articles more\ncarefully.\nResponse: We apologize for the errors. They are now corrected.\n8. Please describe the details in the data availability.\nResponse: We have deposited datasets, including RNA sequencing (RNA-seq) and\nmass spectrometry data, in public repositories to ensure accessibility. Detailed\ninformation is provided in the Data Availability section of the manuscript. These datasets\nare now fully available for reviewer access.\nReviewer #2\n1. K5SMAD7 model indicates overexpression in other stratified epithelia, not only skin.\nthis may contribute to observed differences in the weight, instead authors claim and\noveremphasize skin specific expression.\nResponse: The reviewer has a valid point. Indeed, we did not know if the system effect\nwas mainly from epidermal Smad7 overexpression or from other epithelial tissues as we\noriginally published (He et al., 2002). That was our rationale for topical Smad7-based\ntreatment to assess the direct and local effect of Smad7 on diabetic wounds. We have\nmade this clearer (Result section).\n2. The use of adequate controls is questionable throughout the manuscript: vehicle does\nnot represent ideal control, scramble protein would be more appropriate. Also, WT mouse\nshould be included as a control for K5SMAD7.\nResponse: The specificity of Smad7-based protein was previously confirmed by using\nTat-Cre, an irrelevant Tat protein as a control (Han et al., 2013). Therefore, the current\nstudy using vehicle as control is for a translational design that assesses a protein-specific\nefficacy and potential toxicity, which would be conducted the same way in future clinical\ntrials. Scramble protein is not appropriate in this setting because each protein/peptide has\nits own potential to be an immunogen or have other side effect on open wounds. Using\nK5.Smad7 transgenic mice is another reason to rule out a non-specific effect of Tat-PYC-\nSmad7. In addition, to rule out a placebo effect from vehicle (would be required for clinical\ntrial as well), we tested two vehicle formulations in diabetic pig wounds: HPC (F42, 1%\nKlucel HF, Ashland) with PBS and 20% or 30% glycerol, and HEC (1% Natrosol 250HX,\nAshland) with PBS and 30% glycerol, neither affected wound healing. We have made\nthese points clearer in the revised manuscript. We agree with the reviewer that WT mice\nshould be included in studies using K5.Smad7 model. We had these mice in the\nexperiments but since they behaved the same as WT/db mice, the data were largely\nredundant. In response to reviewer’s request, we have added data from WT mice in Fig\n1B-E.\n3. The authors should comment on the use of animal models to evaluate effect of human\nrecombinant protein.\nResponse: The amino acid sequence encoding the PY domain and C-terminus of the\nhuman Smad7 protein has 99% homology to mouse and pig Smad7, thus animal models\nused in this study will allow us to test its function without causing species-mismatch\nassociated immunogenic response. We have added this rationale to the Introduction. In\naddition, we performed an anti-drug antibody (ADA) assay using plasma samples\ncollected from pigs at the end of the experiment. The results did not detect anti-Smad7\nantibodies, indicating that the topical application of Tat-PYC-Smad7 did not elicit a\ndetectable immune response within the 16-day treatment period. This new result has\nbeen added to Sup Fig. 6C.\n4. In Figure 1A: include K5.SMAD7 mouse in F1A along with K5.SMAD7/db/db; Figure\n1N: is missing x axis label.\nResponse: We apologize for the sloppy mistakes and have corrected them in the revised\nmanuscript.\n5. Please specify the age of mice used, K5SMAD7 controls shown are in telogen while\ndb/db mice are in anagen phase. This is of significant concern as the healing outcomes\nare influenced.\nResponse: We used 8-12 weeks old mice (littermates), including db/db mice, throughout\nthe study, and mice within each experiment were of the same or very similar ages (+/- 4\ndays), thus hair follicles in all of these mice are in the telogen phase (not anagen phase)\nto ensure consistency. The patchy hair loss among K5.Smad7 mice is due to the\nphenotype of this strain with abnormal hair cycles (Han et al, Dev Cell, 2006). We agree\nwith the reviewer that the hair cycle difference could be a compounding factor for the\nhealing outcome, which is another reason we also tested topical treatment on mice with\nthe same hair cycle. We have added this clarification and rationale to the revised Result\nsection.\n6. TAT-SMAD7 has less effect on peripheral immune response compared to Regranex\nused as a control, but further characterization of systemic effect, such as circulating\ncytokine levels, would need to be demonstrated to claim that TAT-SMAD7 has only local\neffects. This and multiple other overstatements should be revised.\nResponse: Following the reviewer’s suggestion, we collected the plasma samples from\ndb/db mice treated with Regranex, Tat-PYC-Smad7 or vehicle control and conducted a\ncytokine array to detect circulating cytokine levels and further explore the potential\nsystemic effects. We found that Tat-PYC-Smad7 has no significant effects on systemic\ncytokine production. In contrast, Regranex had systemic effects on upregulating pro-\nangiogenic factors such as Angiopoietin 2, chemokines such as CXCL16/CXCL5, LDL R\nand IGFBP3 (Sup Fig.5B and C). Angiopoietin-2 (Ang-2) is a pro-angiogenic factor that\npromotes vascular leakage, which has been involved in chronic inflammation and fibrosis\n(Saharinen et al., 2017). Chemokines such as CXCL16 and CXCL5 recruit immune cells\nto inflamed tissues, facilitating fibrotic processes (Korbecki et al., 2021). In addition, LDL\nreceptor increases oxLDL accumulation in vascular-rich tissues, activating macrophages\nand endothelial cells to produce pro-inflammatory cytokines and reactive oxygen species,\npromoting fibrosis (Zhang et al., 2016). The results of this test have been included in Sup\nFig.5B and C. Additionally, we added data that diabetic pig serum treated with Tat-PYC-\nSmad7 in wounds did not show detectable systemic Tat-PYC-Smad7 (Sup Fig. 6B).\nThese data further support the local effects of Tat-PYC-Smad7.\n7. Figure 2D: show corresponding D1 and D16, as comparison across conditions at a\nsingle time point other than day 1 is less meaningful. In Figure 2E show % of wound\nclosure for each condition, normalized to initial wound size.\nResponse: In the pig experiment, we could only collect tissue samples at the endpoint of\nthe study, which prevented us from obtaining histological images from corresponding D1\nfor comparison with D16. Therefore, gross image quantifications are based on\ncomparison with the initial wound sizes consistent across groups. Fig.2E presents the\nhealing rate for each condition, as assessed by histological evaluation. We quantified the\nwound length on day 16 through histological evaluation and calculated the percentage of\nremaining wound length relative to the initial wound length calculated by the punch\ndiameter. These new quantifications are added to Fig. 2F and G.\n8. Figure 3: more Ki67 indicates more proliferating cells – co-staining is required to\nconclude the effect on keratinocytes. Additional staining of fibroblasts should be\nincluded, at minimum.\nResponse: In Fig 3A, we used keratin 14(K14) to stain keratinocytes and αSMA as the\nmarker for fibroblasts. We quantified the Ki67-positive cells only in the migrating tongue\nof the epidermis positive for K14 staining. In the revised figure, we changed Y-axis as %\nKi67+/K14+ keratinocyte/mm2 to be clearer.\n9. The reproducibility is also an issue; mouse experiments have been replicated twice\nonly. Porcine model requires more wounds.\nResponse: We recognize the importance of reproducibility and have made efforts to\nensure consistency across our experiments. Breeding K5.Smad7 db/db mice is\nchallenging due to the infertility of homozygous db/db individuals. Therefore, generating\nK5.Smad7 db/db mice required crossing K5.Smad7 transgenic mice with heterozygous\ndb (db/+) mice. Therefore, the mouse experiments using transgenic mice were bred with\nmultiple litters and repeated twice, and experiments were performed using littermate\ncontrols. We have merged the results from two independent experiments in Fig. 1E in\nthe revised manuscript, which includes quantification from 6-15 mice per group. For\nexperiments involving db/db mice receiving protein treatments, we have added\nindependent repeats in Fig. 1K and Sup Fig. 2.\nWe acknowledge the limitation of few pigs with limited wounds per pig. The Yucatan\nmini-pigs (27–30 kg) were first chemically treated to induce diabetes (mimicking Type I\ndiabetes in humans), then being screened to determine which ones had sustained high\nblood glucose (because not all of them did) but were not severely ill (due to diabetic\ncomplications) to be used for experiments. Based on IACUC allowable criteria, the\nnumber of wounds is limited to 12 per pig, each 2cm in diameter. Because the clinical\ncare from the clinical veterinary team for these pigs and the associated costs are more\nextensive than in normal pigs, and the latency from waiting for pigs to be ready to do\nexperiments was about a year, adding more pigs are both time- and cost-prohibitive.\nTaken together, pig wounds can only be used to validate the relevance to human diabetic\nwounds but are limited for in-depth research. We have acknowledged this limitation and\nthe rationale for a complementary approach including both porcine and mouse models\nfor the study in the Introduction.\n10. Details such as type of dressing used for porcine wounds are not included; what\ntype of 3M film was used for murine wounds?\nResponse: For the porcine wounds, we used 3M Tegaderm film to cover each wound.\nThe specific details were put into the Supplemental Methods based on the journal’s\nrequest.\n11. Inhibition of NETosis is novel, however it lacks mechanistic insights.\nResponse: We thank the reviewer for recognizing the novelty of our findings regarding\nthe inhibition of NETosis by Tat-PYC-Smad7. The reviewer’s comment also highlights the\nchallenge of NETosis research field, i.e., the lack of knowledge about which signaling\npathways regulate NETosis, which would give us the leads to dive into Smad7-associated\nmechanisms. Therefore, in the revised manuscript, we first examined if the observed\neffect is a physiological or pharmacology mechanism. We knocked down endogenous\nSmad7 in dHL60 neutrophils and found that PMA-induced and high glucose-induced\nNETosis was increased with Smad7 knockdown (Fig. 5H-J), suggesting this is Smad7-\nspecific mechanism. Second, mechanistically, Smad7 effect on NETosis is not due to\nblockade of TGFβ signaling (Sup Fig. 13). Instead, Tat-PYC-Smad7 blocks neutrophil\nhistone H3 citrullination (Fig. 6A-D) and reduces MPO activity (Fig. 6F). MPO is essential\nfor NETosis, as it translocates into the nucleus to promote chromatin unfolding and\ndecondensation (Dufrane et al., 2006, Thiam et al., 2020). In our revision, we performed\nimmunostaining and confocal imaging to examine the localization of MPO within\nneutrophil granules versus the nucleus following PMA stimulation in Tat-PYC-Smad7-\ntreated dHL60 cells. Our results showed that Tat-PYC-Smad7 localized to the nucleus\nof dHL60 cells, while MPO staining appeared consistent with granule localization\nfollowing PMA stimulation (Fig. 6G). Our data also showed that Tat-PYC-Smad7-treated\ncells retained MPO within granule-like structures (Sup Fig. 15), suggesting that Tat-PYC-\nSmad7 may interfere with the large-scale release of granule components, i.e., an early\nand critical step during NETosis. These new findings have been added to Fig. 6G, Sup\nTable 1, Sup Fig. 15 and Fig. 5F-J. Taken together, the mechanisms of Tat-PYC-Smad7\non neutrophil NETosis could involve reduced MPO enzymatic activity (Fig. 6F),\nthe reduction of MPO escape from the granules (Fig. 6G) which otherwise would potentially\ninitiate NETosis, as well as Tat-PYC-Smad7/MPO nuclear colocalization (Fig. 6G) that\npotentially reduce MPO-induced chromatin decondensation during NETosis. We have\nrevised the manuscript accordingly to address the reviewer’s concerns. Finally, in our\nrevised discussion, we pointed out that our current findings warrant future independent\nstudies for NETosis mechanisms and the mechanisms of Smad7 in this process at the\ncellular and molecular levels.\n12. Figure S1: evidence of dermabond is visualy noticable, but selectively on db/db\nwounds. This confounds wound healing.\nResponse: We did not use dermabond during the wound experiment. The \"dermabond-\nlike\" appearance observed in previous Sup. Fig 1 (Sup. Fig 3 in the revised manuscript)\nin the Regranex-treated groups is actually due to the residual marks left by the application\nof Regranex, which is an adhesive gel with high viscidity. In the revised manuscript, we\nhave added this explanation in figure legend to avoid confusion.\n13. Figure S2: Requires detailed labeling to indicate what morphology is being referred\nto as foam-like, as all three conditions show F4/80+ cells.\nResponse: We appreciate the reviewer’s suggestion. We have revised the label for\nF4/80⁺ foam-like cells in previous Figure S2 (current Sup Fig. 4A in the revision). In\naddition, since the staining intensity and cell size of F4/80⁺ foam-like cells are distinct\nfrom other F4/80⁺ myeloid cell populations, we applied a mask to identify foam-like cells\nand defined it using HSV thresholding (H: 4–36, S: 35–101, V: 158–218) to set the signal\nintensity. Additionally, a minimum object size threshold of 15 pixels was used to define\ncell size, utilizing the Count & Measure function in CellSense software (Olympus, version\n2.3). This approach objectively distinguishes foam-like cells from the general F4/80⁺\nmyeloid cell population, as shown in Sup Fig. 4B. Based on this method, we quantified\nF4/80⁺ foam-like cell objects in vehicle-, Regranex-, and Tat-PYC-Smad7-treated db/db\nwounds, as presented in Sup Fig. 4C. We hope this revision sufficiently addresses the\nreviewer’s concern.\n14. Figure S5: The authors state that ECM remodeling is reflected in CD31 staining for\nendothelial cells, this is not correct.\nResponse: We have corrected the figure legend for this careless mistake. CD31 staining\nreflects angiogenesis, not ECM remodeling. This correction is now in the current Sup\nFig.7.\nReviewer #3:\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports.\nThis is part of the Nature Communications initiative to facilitate training in peer review and\nto provide appropriate recognition for Early Career Researchers who co-review\nmanuscripts.\nResponse: We are glad our manuscript provided this opportunity to the reviewer.\nReviewer #4:\nAlthough good efficacy has been shown in animal models, the safety, efficacy, and\nfeasibility of TAT-PYC-SMAD7 in actual clinical applications still require further validation,\nas there may be challenges in translation. While the study reveals the role of TAT-PYC-\nSMAD7 in inhibiting neutrophil activity and NETosis, the exploration of its specific\nmolecular mechanisms remains insufficient and may require more experimental data to\nsupport. Despite the use of various animal models, the relatively limited sample size may\naffect the statistical significance and generalizability of the results, necessitating further\nexpansion of the sample size for validation.\nResponse: We hope that addition of substantial amount of data from animal experiments\nand mechanistic studies of NETosis in the revised manuscript have addressed the\nreviewer’s concerns. These have been highlighted in the revised manuscript and\nspecifically addressed in responses to other reviewers.\nReviewer #5\n1. The data on TAT-PYC-Smad7 inhibiting PMA-induced NETosis is exciting. To connect\nthis observation back to diabetes, it would be important for the authors to validate the\nimpact of Smad7 on NETosis in more physiologically relevant situations such as when\nNETosis is induced with high glucose. It would also be important to show that expressing\nSmad7 in dHL60 cells prevents NETosis to rule out a potential side effect of the TAT-PYC-\nSmad7 biologics (e.g. smaller size thus higher delivery in the nucleus than endogenous\nSmad7).\nResponse: We appreciate the reviewer’s suggestion and have conducted additional\nexperiments in which NETosis was induced by high-glucose conditions. Our results\nconfirm that Tat-PYC-Smad7 effectively inhibits NETosis under these conditions, further\nsupporting its relevance to diabetic wound healing. As the reviewer suggested, we also\nperformed Smad7 knockdown in dHL60 cells using siRNA targeting Smad7. We found\nthat reducing endogenous Smad7 expression significantly increased the susceptibility of\ndHL60 cells to both PMA- and high-glucose-induced NETosis. Because endogenous\nSmad7 is a nuclear protein, our finding indicates that the effect of Tat-PYC-Smad7 on\nNETosis is specifically linked to the functions of Smad7 . These results and relevant\nmethods are present in Fig. 5F-J and Supplementary Methods.\n2. Because NETs are described as networks of DNA with nuclear/cytotoxic proteins, the\nauthors should demonstrate that cytotoxic green labels NETs by showing that it\ncolocalizes with citH3 neutrophil elastase or MPO. The authors could immunostaining for\nthese markers at the end of the live imaging period. It would also be important to show\nZoomed images of figure 6D – at this resolution, readers cannot evaluate whether the\ncytotoxic green signal is extracellular (e.g. occupies a larger area than the typical cell\narea).\nResponse: We thank the reviewer for the constructive advice. We have performed\nimmunostaining for citH3, and MPO with DNA cytotoxic green labels and confirmed the\ncolocalization with the cytotoxic green signal induced by PMA (Sup Fig. 12). Additionally,\nwe have included zoomed images (Fig.5C in the revised manuscript) to provide a clearer\nview, confirming that the cytotoxic signal is extracellular and occupies a larger area than\nthe typical cell size. These data are added in the revised manuscript.\n3. Further, the authors propose that Smad7 directly binds to MPO in the nucleus thus\npreventing its function in NETosis. Although MPO was shown to be important for NETosis,\nit is still not clear how it mediates the process – for instance, MPO enzymatic activities\nare not required for NETosis (https://doi.org/10.1083/jcb.201006052). Was the IP in figure\n6H done with the nuclear fraction to show that MPO-Smad7 interaction occurs in the\nnucleus? Did the author exclude the possibility that TAT-PYC-Smad could enter neutrophil\ngranules and prevent MPO from accessing the nucleus?\nImmunostaining and confocal imaging could be used to assess MPO nuclear vs granules\nlocalization after PMA stimulation of TAT-PYC-Smad7 treated dHL60 cells.\nResponse: We appreciate the reviewer’s insightful suggestions. The IP was performed\nusing total cell lysates because Tat-PYC-Smad7 can travel between the cytoplasm and\nthe nucleus similar to endogenous Smad7. Following the reviewer’s advice, we conducted\nimmunostaining and confocal imaging to examine MPO localization in neutrophil granules\nversus the nucleus following PMA stimulation in Tat-PYC-Smad7-treated dHL60 cells. We\nfound that without Tat-PYC-Smad7, MPO had more nuclear localization after PMA\nstimulation (Sup Fig. 15). In contract, MPO is largely located in granules with the\npresence of Tat-PYC-Smad7 treatment. Confocal microscopy further revealed that\nalthough colocalization of Tat-PYC-Smad7 with MPO occurred in nuclei of some dHL60\ncells (yellow arrows in Fig. 6G), MPO positive granules did not have colocalization with\nTat-PYC-Smad7 (Fig. 6G). Therefore, our data suggest two potential mechanisms of\nTat-PYC-Smad7-mediated NETosis reduction. First, Tat-PYC-Smad7 treated cells\nretained MPO within granule-like structures (Sup Fig. 15), raising the possibility that Tat-\nPYC-Smad7 may interfere with the large-scale release of granule components — an early\nand critical step during NETosis. Second, Tat-PYC-Smad7 could directly interact with\nMPO once MPO enters the nucleus. Our data warrants future studies for the molecular\nmechanisms governing granule content release during NETosis, and if and how this\nprocess involves positive feedback loops where initial escape of granule contents\nfacilitates further release of NET components and promotes chromatin decondensation.\nAs the reviewer pointed out, the exact mechanisms by which MPO contributes to\nNETosis are largely unknown and would warrant independent studies beyond the scope\nof our current study. Given that MPO appears to have less escape from the granule\ncomponent and NET formation with the presence of Tat-PYC-Smad7 treatment, it is\npossible that Tat-PYC-Smad7 also targets an upstream step in NETosis, with the MPO\ninteraction contributing to — but not solely accounting for — its inhibitory effects.\nWe have added the new imaging data into Fig. 6G and Sup Fig. 15 and revised the\nDiscussion to soften our interpretation of the MPO–Tat-PYC-Smad7 interaction and\nemphasize the need for future studies to dissect the detailed mechanisms underlying\nthese observations. We appreciate the reviewer’s suggestions, which have helped us\nclarify and refine the mechanistic interpretation of our findings.\n4. The authors show that TAT-PYC-Smad7 prevents citrullination of H3 in PMA treated\ndHL60 cells without impacting the expression level of PAD4. This is an interesting dataset\nthat suggests that Smad7 is important for PAD4 enzymatic activity. First, for reproducibility\npurposes, the authors should show and quantify repeats of the western blots shown in\nfigure 6 F and G. In vitro experiments testing the impact of smad7 on PAD4 citrullination\nactivity would be important to clarify how Smad7 prevents H3 citrullination and NETosis,\npotentially independently of Smad7-MPO interaction.\nResponse: As the reviewer recommended, we have added data for western blot repeats\nand quantified the results which are presented in the revised Fig. 6B and D. We sought\nto investigate whether Tat-PYC-Smad7 directly modulates PAD4 enzymatic activity using\na commercially available PAD4 inhibitor assay. However, all commercial PAD4 activity kits\ncontain chemicals (not present in normal cells) interfering with the conformation of Tat-\nPYC-Smad7 protein. As a result, within the technical limitations of the current assay\nsystems, we were unable to directly assess the impact of Tat-PYC-Smad7 on PAD4\nenzymatic activity. We plan to explore this in future investigations when alternative assay\nmethods.\n5. Finally, the author proposes that Tat-PYC-Smad7 prevents neutrophils degranulation.\nCould the author please indicate which data supports this claim? The decreased release\nof neutrophil elastase could also be due to the defects in NETosis since cells release\nneutrophil elastase during NETosis.\nResponse: We agree that the reduced release of neutrophil elastase is more likely due\nto impaired NETosis rather than direct inhibition of degranulation. We have revised the\nmanuscript to reflect this and removed the statement regarding the effects on neutrophil\ndegranulation.\n6. This reviewer appreciates the difficulties of experimenting with animals. However, the\nsample size of the pig dataset is very low. And some of the analysis is done with only Pig\n1 in the main figure (Pig 2 and 3 are pulled in Sup fig.9 which shows a broad distribution)\nwhere the authors perform statistical test (t-test) on samples with 4 data points. This low\nsample size makes the t-test very unreliable. For example, in “figure 3, D)- TAT-PYC-\nSmad7, 4ug” one data point is much higher than the 3 other data points which pulls the\nmean % CD31+ area up – in such a small sample size, an outlier has a strong impact.\nAnother example of how the small sample size impacts the conclusion of the study is in\nfigure 5 D where the distribution of the %Ly6G+CitH3+ cells/area shows two populations\nwhich alters the mean. This data set cannot be tested with a t-test which assumes a\ngaussian distribution of the sample. Thus, the in vivo data (figure 3-C, B, D Fig 5 D and\nE) requires more data points for the statistical tests used here and the conclusions of this\nstudy to be meaningful. The authors should also show the standard deviation of their\ndistribution instead of the standard error of the mean (SEM), which does not allow readers\nto appreciate the heterogeneity of the population.\nResponse: We acknowledge the reviewer’s concerns regarding the sample size in the\npig experiments. Given that the results of IHC/MSI quantification showed similar trends\nfor the three pigs, we merged the data from the three pigs for quantification to improve\nthe statistical integrity. Former Fig. 3D, E and Fig. 5E (Fig. 5B in the revised manuscript),\nafter merging the data from pig 1-3, we used ROUT method (Q = 1%) to identify the\noutliers of the data. After eliminating the outliers from data, we used Shapiro-Wilk\nnormality test to detect the normality of the data. For the data sets fit the Gaussian\ndistribution, we used a two-trailed unpaired t-test for pairwise comparison between two\ngroups. For data that do not fit Gaussian distribution, we used the Mann-Whitney test for\npairwise comparison between two groups. For data from Fig. 5D (Fig. 5A in the revised\nmanuscript), we did a new set of staining using samples from another independent\nexperiment. We merged two data sets and did the same statistical processing as\ndescribed above. We have revised the manuscript to clearly state the limitations regarding\nsample size for pig study and have updated the figures to present SD rather than SEM,\nproviding a more accurate reflection of data variability.\n7. A big picture question that the authors might want to discuss is the potential side effects\nof Tat-PYC-Smad7 biologics. If this biologic prevents neutrophil functions, and MPO\nactivity - which is important for host defense against microbes, will the application of Tat-\nPYC-Smad7 enhance pathogen propagation at the wound site?\nResponse: We appreciate the reviewer’s important question regarding the potential side\neffects of Tat-PYC-Smad7, particularly its impact on neutrophil function and host defense\nagainst infections. In our animal models, we did not observe an increase in infection rates\nfollowing Tat-PYC-Smad7 treatment in db/db mouse studies or pig studies. Additionally,\nour data showed reduced infiltrating Ly6G+ neutrophils on day 11 but not on day 9 post-\ntreatment (Fig. 4E and F). This suggests that Tat-PYC-Smad7 does not affect neutrophil\nrecruitment during early stage of wound healing when host defense is more prominent.\nFurthermore, Tat-PYC-Smad7 treatment did not trigger any changes in CBC parameters\nor little/effects on systemic cytokine levels (Sup Fig. 5), indicating that its functional\nimpact on neutrophils may be localized to the wound site and is likely limited to later\nstages of wound healing.\nNeverthelss, we agree with the reviewer that the effect of Tat-PYC-Smad7 on host\ndefense against microbes needs to be more comprehensively studied in the future. We\nhave revised the discussion section to highlight this limitation and point to future studies\nto directly evaluate the safety and potential effects of Tat-PYC-Smad7 on neutrophil-\nmediated antimicrobial responses."}, {"round": 2, "reviewer_comments": {"reviewer_1": "Comments： While we acknowledge the comprehensive revisions made to this manuscript, several critical issues remain unresolved that affect the study's validity and interpretation. First, concerning sample size and model validation: The persistent issue of small sample sizes with apparent individual differences in Figures 1B and 1C significantly undermines experimental reproducibility. We note the authors' explanation regarding model construction difficulties, but these technical challenges do not alleviate concerns about statistical power(In Fig1B and C, there are small samples of different numbers in the same group. Originally, such small samples lack repeatability.The observed individual variations necessitate re-evaluation of the model's reliability, as similarly highlighted by Reviewers 2 and 4. Second, regarding mechanistic elucidation: The proposed regulation of MPO by Smad7 lacks comprehensive experimental validation. While binding interactions are demonstrated, the precise regulatory mechanism within the NETosis pathway (both upstream and downstream) remains largely speculative (the author only discussed binding, and how Smad7 regulation /MPO is affected in the upstream and downstream parts of NETosis is still mainly speculative). As Reviewer 5 appropriately noted, the potential cytoplasmic regulation remains unverified, representing a significant gap in the mechanistic framework. Finally, we hope that after this round of revision, the article can achieve further improvement. Question 1: It has been completely solved Question 2:We recognize the relevant elaboration, but still feel that it lacks strong persuasiveness Question 3: This part has been corrected Question 4-1：Our concerns about the fourth part are not unnecessary. First of all, the diabetic mouse model should have elevated blood glucose concentration for a long time (for more than 4 weeks). The significance in Fig1B and Fig1C is to demonstrate the role of Smad7 in wound healing. However, we found that in the K5Smad7/db/db mice, there was a group of mice whose blood glucose levels fluctuated abnormally. Should this mouse be regarded as having individual differences? And just as several other reviewers were concerned, we believe that a small sample size makes it difficult to determine causal relationships and is accidental. We do not deny that Smad7 has a good effect in regulating NETosis, but blood glucose level is also an important indicator of wound healing, even though it has been verified in later articles. However, it is hoped that this phenomenon of blood sugar fluctuations can be explained to support the errors caused by too few animal experiments. Question 4-2：Furthermore, regarding the animal part, the wound healing images presented by the db/db group in Fig1D show almost no visual difference, but there is a significant wound healing effect during the statistical process. We believe that, like other animal experiments, a scale or control substances should be added. Therefore, it is hoped that the author can adjust the zoom ratio of the pictures and place the scale ruler to enhance the readability of the article. Question 5: It has already answered our confusion very well Question 6:Relevant changes were seen in the text and we think they are relatively reasonable Question 7:The existing problem parts have been corrected Question 8:It has been revised.", "reviewer_2": "The authors have improved mechanistic aspects focused on NETosis, however multiple issues remain. -Fig 1L, not clear what blue lines represent, please clarify. -Fig 2D, the area selected to show epidermal wound edge in the Smad7 treated wounds is questionable as epidermis appears as crust, quantification of K14 staining would be more appropriate than h&e -quantification of CD31/angiogenesis in porcine wounds is questionable as it is expressed as \"objects per migrating epithelium\" (Fig 3). Detailed CD31 quantification requires area of cd31+ blood vessels per mm2 of the wound bed, regardless of epithelialization. -a-sma is diminished in Smad7-treated murine wounds (fig 3a), while this is not surprising it may impact the quality of scare tissue, please quantify and discuss. -the area selected as representative for elastic fibers in a pig wound.day 16 is questionable as well, as it does not focus on the wound bed, it shoes unwounded area. -significant number of genes regulated at the later time point, day 11, suggests regulation of processes beyond the neutrophil function, the most active phase of neutrophils during wound healing is between days 3-5, please comment. -RNAseq data and manuscript overall would be significantly improved by comparative transcriptional analyses from generated RNAseq post-Smad7 treatment and the publicly available datasets from human diabetic foot ulcers. -In fig 5b, CitH3/MPO staining enlargement of vehicle and tat-pyc-Smad7 treated porcine wounds is mostly focused on the \"crust\" area which often results in unspecific staining in the porcine wound model. It is is not clear why authors do not focus on the actual wound bed area with significant CitH3 signal. Similarly in murine wounds area above epidermis is zoomed in for the vehicle treated wounds, while wound bed is zoomed in for the tat-pyc-Smad7 treated wounds (Fig 5a) -the manuscript would be significantly improved by comparative transcriptional analyses from the generated RNAseq post-Smad7 treatment with the public datasets from human diabetic foot ulcers.", "reviewer_3": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_5": "In this revised version of their manuscript, Ke et al., significantly deepened their demonstration of Smad7 expression preventing PMA-induced NETosis. They expanded their study to show that smad 7 also prevents high glucose-induced NETosis which is extremely relevant to their hypothesis that smad 7 expression facilitates the healing of diabetic wounds partially through inhibition of NETosis. This is a nice and extensive study that is ready for publication. This reviewer recommends the minor corrections below: 1. While the role of smad7 in preventing NETosis is convincing, the molecular mechanisms lack strong supportive data. The confocal images in Sup figure 11, 12, 15 have limited resolution / are blurry, which makes it hard to assess MPO granular versus nuclear localization. The data currently in the manuscript shows that MPO colocalize with DNA and smad7-HA (Figure 6, Zoom), but whether this localization occurs inside an intact nucleus or after DNA is released from the nucleus is not clear. I strongly suggest the authors clarify this in the manuscript so that follow up studies can rigorously address the question of when and where Smad 7 /MPO interaction occurs. 2. Figure 5I: It could be helpful to readers to see zoomed images as in Figure 5F. 3. Figure 6B: Please indicate on the figure panel that B quantifies CitH3. How is B normalized? From panel 6A, it looks like CitH3 is highest at 30 min but the graph shows comparable values. The authors could consider normalizing relative to the first time point to show how CitH3 levels change over time after PMA stimulation. 4. Figure 6C: Interesting to note that no CitH3 was detected in cells treated with 3 ug of TAT-PYC-Smad7 while this amount of the biologics minimally impairs NETosis based on figure 5. 5. Figure 6E: How long after PMA stimulation? does it match with a limited co-localization observed in 6G?"}, "author_response": "Point-by-Point Response to Reviewers’ Comments\nWe sincerely appreciate that the editorial board has given us an opportunity to revise and\nresubmit our manuscript. We are grateful for the thoughtful and constructive comments\nfrom the reviewers. We have added more data to address reviewers’ concerns and\nrevised our manuscript based on reviewers’ comments and new data. Responses to\nreviewer’s comments are provided below explaining how the concerns are addressed\nexperimentally and/or conceptually. In the revised manuscript, contents directly\naddressing reviewers’ concerns are highlighted in yellow. We hope that our responses\nhave addressed all the reviewers’ concerns, and the revised manuscript is strengthened\nsignificantly.\nCritiques – Responses\nReviewer #1\n1. First, concerning sample size and model validation: The persistent issue of small\nsample sizes with apparent individual differences in Figures 1B and 1C significantly\nundermines experimental reproducibility. We note the authors' explanation regarding\nmodel construction difficulties, but these technical challenges do not alleviate concerns\nabout statistical power (In Fig1B and C, there are small samples of different numbers in\nthe same group. Originally, such small samples lack repeatability. The observed individual\nvariations necessitate re-evaluation of the model's reliability, as similarly highlighted by\nReviewers 2 and 4. \nResponse: Fig. 1B and 1C consist of 4 genotypes in which most important comparisons\nare between db/db and Smad7/db/db mice. We could not control equal distributions of\nsex and genotypes during breeding to generate these bigenic mice (and it is noteworthy\nthat both strains are poor breeders). Therefore, previous data were from female mice\nbecause K5.Smad7 genotype only had one male mouse. To make the data more\ntransparent, we included data from pooled sexes in the revised Fig. 1B and C and\nincluded genotypes and sex in Sup Table 1. Each mouse’s genotype, sex, weight and\nglucose can be found in the data source file. As you can see below, the revised figures\nwith more mice did not change the conclusion. Specifically, K5.Smad7/db/db mice (n =\n10) showed reduced weight and glucose levels compared to db/db mice (n = 16), with a\nsubset of K5.Smad7/db/db mice even displaying glucose levels within the normal range.\nThis observation prompted us to include Tat-PYC-Smad7 topical treatment to the wounds\nPrevious Fig. 1B, 1C Revised Fig. 1B, 1C\nin db/db mice to directly test whether the effect of Smad7 is systemic or primarily local in\npromoting wound healing. Additionally, Tat-PYC-Smad7 treatment to pig wounds included\nvehicle controls in each pig in which systemic glucose and weight are the same. We hope\nthese three complementary experimental systems could convince the reviewer that\nSmad7 effects are largely at the local/wound site.\n2. The proposed regulation of MPO by Smad7 lacks comprehensive experimental\nvalidation. While binding interactions are demonstrated, the precise regulatory\nmechanism within the NETosis pathway (both upstream and downstream) remains largely\nspeculative (the author only discussed binding, and how Smad7 regulation/MPO is\naffected in the upstream and downstream parts of NETosis is still mainly speculative). As\nReviewer 5 appropriately noted, the potential cytoplasmic regulation remains unverified,\nrepresenting a significant gap in the mechanistic framework.\nResponse: There are still significant gaps in the field’s understanding of the basic Netosis\nmechanism itself (including both up- and downstream steps), which makes it challenging\nto assess the step at which Smad7 acts. This fact does not diminish the novelty of the\nobservation that Smad7 inhibits NETosis. However, to further address the reviewer’s\nconcern and add mechanistic insight, we performed a new experiment including co-\nstaining of Lamin B1 (a nuclear membrane marker), MPO, HA, and DAPI in dHL60 cells\ntreated with Tat-PYC-Smad7 or vehicle control at early stages during PMA-induced\nNETosis, to examine whether MPO colocalization with DNA and Smad7-HA occurs within\nan intact nucleus or only after DNA release. The data have been added to the manuscript\nas revised Fig. 6G. We find that during dHL60 cell stimulation with PMA, MPO enter the\nnucleus in control cells by 1 hour, but it is often excluded from the nucleus in Tat-PYC-\nSmad7-treated cells at the same time point. When colocalization with DNA and Smad7-\nHA does occur, it is often detected within cells that retain an intact Lamin B1 ring. We\ndiscuss these results and their mechanistic implications, but we have also tried to be\ncareful to avoid overinterpretation. We hope these data provide more insight into potential\nmechanisms.\nAs Reviewer 5 noted, the precise mechanisms by which MPO contributes to NETosis\nremain largely unknown and would warrant independent studies beyond the scope of this\nwork. Nevertheless, our study provides a novel mechanistic entry point by identifying\nSmad7-MPO interactions and a role for Smad7 in opposing nuclear entry of MPO during\nearly stages of NETosis. These observations could guide future investigations as the field\nadvances. We have modified our Discussion section to describe these results and to\nmake it clear that there are important mechanistic gaps that will need to be resolved in\nfuture studies.\n3. Cited reviewer’s question and our response from rebuttal of NCOMMS-24-47156-R1:\n“Question 2: A previous study has reported the feasibility of using exogenous Smad7\nbelow an oncogenic level to alleviate skin inflammation and wound healing defects\nassociated with excessive activation of TGF-β and NF-κB. In my view, the ideas of this\nstudy coincide to some extent with those of this previous study. In addition, Tat-PYC-\nSmad7 reduced neutrophil infiltration and attenuated TGF-β and NFκB signaling\npathways were also reported (PMID: 34610386). It is necessary to add and highlight the\ndiscussion about the innovation points of this study.\nResponse: We agree with the reviewer’s comment that we should more clearly delineate\nconceptual innovations and novel findings of the current study compared to our previously\npublished studies. In the revised discussion, we have clarified the novelty of the current\nstudy: in addition to providing direct evidence (compared to previous correlative studies)\nthat Smad7-based therapeutic effects target TGFβ and NFκB in two diabetic wound\nmodels, we identified that the effect of Smad7 on neutrophil activity, specifically on\nNETosis, which is independent of TGFβ signaling. This effect is difficult to capture in other\nexperimental model systems in which neutrophil infiltrations are often transient.”\nReviewer’s comment regarding the cited content in Question 2: We recognize the relevant\nelaboration but still feel that it lacks strong persuasiveness.\nResponse: Unlike our earlier proof-of-concept work, here we directly test Smad7 in bona\nfide diabetic wound models using both preclinical mouse and pig systems, establishing\nits therapeutic potential in clinically relevant contexts. Importantly, we identify a new\nmechanism, the suppression of excessive NETosis, which extends beyond previously\nreported effects and provides translationally significant insights into diabetic wound\npathology. We have revised the Introduction and Discussion to emphasize these\ninnovations more explicitly.\n4. Question 4-1：Our concerns about the fourth part are not unnecessary. First of all,\nthe diabetic mouse model should have elevated blood glucose concentration for a long\ntime (for more than 4 weeks). The significance in Fig1B and Fig1C is to demonstrate the\nrole of Smad7 in wound healing. However, we found that in the K5Smad7/db/db mice,\nthere was a group of mice whose blood glucose levels fluctuated abnormally. Should this\nmouse be regarded as having individual differences? And just as several other reviewers\nwere concerned, we believe that a small sample size makes it difficult to determine\ncausal relationships and is accidental. We do not deny that Smad7 has a good effect in regulating\nNETosis, but blood glucose level is also an important indicator of wound healing,\neven though it has been verified in later articles. However, it is hoped that this phenomenon of\nblood sugar fluctuations can be explained to support the errors caused by too few animal\nexperiments.\nResponse: We would like to clarify that the db/db mice indeed have lifetime high glucose,\nwe only showed the data encompassing our experimental period. As the reviewer noticed,\nK5.Smad7/db/db mice showed significantly lower weight and glucose levels and this\nholds true even with additional pooled mouse data (revised Fig. 1B and 1C). For this\nreason, we generated a Tat-PYC-Smad7 protein to test its treatment efficiency on diabetic\nwound via topical application. We found that topical treatment did not affect glucose or\nbody weight observed in the transgenic model yet still promoted healing, indicating that\nthe Smad7 effect is mainly at the local wound site. As the reviewer indicated, high glucose\nin db/db mice contribute to NETosis, and topical Smad7 in this high glucose condition still\nhas effects. These data do not contradict but rather support reviewer’s point.\n5. Question 4-2：Furthermore, regarding the animal part, the wound healing images\npresented by the db/db group in Fig1D show almost no visual difference, but there is a\nsignificant wound healing effect during the statistical process. We believe that, like other\nanimal experiments, a scale or control substances should be added. Therefore, it is hoped\nthat the author can adjust the zoom ratio of the pictures and place the scale ruler to\nenhance the readability of the article.\nResponse: We greatly appreciate that the reviewer identified the deficiency for our data\npresentation and provided this excellent suggestion. In the revised Fig.1D, we have\ncalibrated and adjusted the zoom ratio, added scale bars, and selected more\nrepresentative images that are consistent with the quantitative wound healing results. We\nbelieve these changes improve the readability of the figures and better reflect the\nstatistical outcomes.\nReviewer #2\nThe authors have improved mechanistic aspects focused on NETosis, however multiple\nissues remain.\n1. Fig 1L, not clear what blue lines represent, please clarify.\nResponse: In Fig. 1L, the blue lines indicate the leading edge of the migrating epidermal\ntongue, identified by reduced collagen density and the absence of hair follicles as\nvisualized by H&E staining. We have added this clarification into the corresponding figure\nlegend.\n2. Fig 2D, the area selected to show epidermal wound edge in the Smad7 treated\nwounds is questionable as epidermis appears as crust, quantification of K14 staining\nwould be more appropriate than h&e\nResponse: In the revised Fig.2D, we have shown the enlarged image with higher\nresolution to show the epidermal wound edge is not the crust. We also added the K14\nstaining of the skin sections to show the consistency of the epidermis shown in H&E.\n3. Quantification of CD31/angiogenesis in porcine wounds is questionable as it is\nexpressed as \"objects per migrating epithelium\" (Fig 3). Detailed CD31 quantification\nrequires area of cd31+ blood vessels per mm2 of the wound bed, regardless of\nepithelialization.\nResponse: We apologize for not describing our quantification clearly. The vessels were\nindeed quantified as per mm2 of the wound bed (see the square boxes) as recommended\nby this reviewer, but we chose the area directly underneath the migrating epithelium.\nBecause angiogenesis at the leading edge is essential for wound closure, providing\nvascular support that directly correlates with re-epithelialization, we simply used the\nmigrating epidermal length as a reference. In the revised manuscript, we have revised\nthe Y-axis from previous “/mm2 migrating epithelium” to “/mm2 wound bed underneath\nmigrating epidermis” to make it clear.\n4. -a-sma is diminished in Smad7-treated murine wounds (fig 3a), while this is not\nsurprising it may impact the quality of scar tissue, please quantify and discuss.\nResponse: We have added the quantification of the a-sma in Vehicle and Tat-PYC-\nSmad7-treated wounds in revised Fig.3A and add the result in the Results and\nDiscussion sections.\n5. The area selected as representative for elastic fibers in a pig wound day 16 is\nquestionable as well, as it does not focus on the wound bed, it shows unwounded area.\nResponse: The representative image of elastic fibers at Day 16 was selected to illustrate\nthe transition zone adjacent to the wound margin, where remodeling of elastic fibers\noccurs as part of re-epithelialization and matrix deposition. Our intent is to highlight\nchanges at the interface between wounded and unwounded tissue, rather than in the\nintact dermis. We have revised the figure legend to specify that the image depicts the\nwound margin area. In addition, we have added images closer to the wound margin area\nto confirm that the observed elastic fiber alterations are consistent across the wound area\nin the revised Fig.3F.\n6. Significant number of genes regulated at the later time point, day 11, suggests\nregulation of processes beyond the neutrophil function, the most active phase of\nneutrophils during wound healing is between days 3-5, please comment.\nResponse: To address the reviewer’s comment, we have added Ly6G staining on days\n3 and 5 of the db/db wounds in the Sup. Fig. 9B. These analyses revealed only sparse\nneutrophil infiltration in the wound beds, which is consistent with previous reports showing\nthat diabetic conditions impair neutrophil chemotaxis and delay their recruitment to the\nwound site (Roy et al., 2022). This explains why a larger number of genes appear\nregulated at the later time point (day 11), when neutrophil activity remains detectable in\ndb/db wounds compared with the more transient peak typically observed between day 3-\n5 in normal wound healing.\n7. RNAseq data and manuscript overall would be significantly improved by comparative\ntranscriptional analyses from generated RNAseq post-Smad7 treatment and the publicly\navailable datasets from human diabetic foot ulcers.\nResponse: We appreciate the reviewer’s thoughtful suggestion. After surveying available\nresources, we found that datasets from interventional clinical trials on diabetic foot ulcers\nare very limited. We identified one project (GSE269483) in the Gene Expression Omnibus\ndatabase that included 10 human samples in a treatment setting; however, the treatment\noutcomes were not disclosed, which prevents meaningful comparison with our study. At\npresent, we are not aware of publicly accessible datasets that would allow us to evaluate\ntreatment effects in a way that could be directly aligned with our Smad7 work. Moreover,\nexisting human diabetic foot ulcer datasets are highly heterogeneous, reflecting\ndifferences in ulcer stage, infection status, anatomical site, diabetes duration, and patient\ncomorbidities. These factors make it challenging to draw parallels with the controlled,\ntime-matched wound healing experiments performed in our mouse model. For these\nreasons, we are not able to include a reliable data-mining analysis at this stage.\n8. In fig 5b, CitH3/MPO staining enlargement of vehicle and tat-pyc-Smad7 treated\nporcine wounds is mostly focused on the \"crust\" area which often results in unspecific\nstaining in the porcine wound model. It is not clear why authors do not focus on the actual\nwound bed area with significant CitH3 signal. Similarly in murine wounds area above\nepidermis is zoomed in for the vehicle treated wounds, while wound bed is zoomed in for\nthe tat-pyc-Smad7 treated wounds (Fig 5a)\nResponse: We focused on the crust/surface region because NETs typically accumulate\nat the wound surface where neutrophils release extracellular traps into the exudate, as\nreported in cutaneous wound studies (Zhu et al., 2021). To ensure specificity, only\nCitH3/MPO/DNA co-localized structures were quantified. For clarity, we have revised Fig.\n5 to include matched regions from both the surface crust and underlying wound bed. We\nhave added this data in the revised Fig. 5A and B.\nReviewer #3:\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports.\nThis is part of the Nature Communications initiative to facilitate training in peer review and\nto provide appropriate recognition for Early Career Researchers who co-review\nmanuscripts.\nResponse: We are glad our manuscript provided this opportunity to the reviewer.\nReviewer #5\n1. While the role of smad7 in preventing NETosis is convincing, the molecular\nmechanisms lack strong supportive data. The confocal images in Sup figure 11, 12, 15\nhave limited resolution / are blurry, which makes it hard to assess MPO granular versus\nnuclear localization. The data currently in the manuscript shows that MPO colocalize with\nDNA and smad7-HA (Figure 6, Zoom), but whether this localization occurs inside an intact\nnucleus or after DNA is released from the nucleus is not clear. I strongly suggest the\nauthors clarify this in the manuscript so that follow up studies can rigorously address the\nquestion of when and where Smad 7 /MPO interaction occurs.\nResponse: We thank the reviewer for pointing out the need to clarify whether MPO\ncolocalization with DNA and Smad7-HA occurs inside an intact nucleus or only after DNA\nrelease. To address this, we performed additional immunofluorescence experiments with\nLamin B1 (a nuclear membrane marker), MPO, HA, and DAPI co-staining.\nThis new data is presented in revised Fig.6G. It shows that in the presence of Tat-\nPYC-Smad7, MPO is mostly excluded from the nucleus at 1 hour. In contrast, we also\nsee cells in which MPO has entered the nucleus, and we observe colocalization of MPO\nwith DNA and Tat-PYC-Smad7-HA within a continuous Lamin B1 ring, indicating that the\nnuclear envelope remains at least largely intact at this stage. While we cannot fully rule\nout subtle nuclear alterations, these results suggest that MPO nuclear entry and its\ncolocalization with DNA and Tat-PYC-Smad7-HA occur prior to overt disruption of the\nnuclear membrane. We have revised the manuscript in the Results and Discussion\nsections to include this clarification.\n2. Figure 5I: It could be helpful to readers to see zoomed images as in Figure 5F.\nResponse: In the revised figure, we have added zoomed-in panels for Figure 5I, similar\nto Figure 5F, to provide clearer visualization and enhance readability for the readers.\n3. Figure 6B: Please indicate on the figure panel that B quantifies CitH3. How is B\nnormalized? From panel 6A, it looks like CitH3 is highest at 30 min but the graph shows\ncomparable values. The authors could consider normalizing relative to the first time point\nto show how CitH3 levels change over time after PMA stimulation.\nResponse: We thank the reviewer for this suggestion. In the revised figure, we have\nindicated in the panel label that Fig.6B quantifies CitH3. The grayscale intensity of protein\nbands was quantified using ImageJ software. Each band was normalized to its\ncorresponding loading control, and the resulting values were further adjusted relative to\nthe highest intensity band, which was set to 1. The statistical analysis was performed\nusing data from three independent experiments. We have described the quantification\nmethod in the supplementary method. The apparent stronger CitH3 signal at 30 min in\nthe blot of Figure 6A appears less distinct in the graph because of this normalization\napproach as the background density in the other two lanes are relatively stronger as well.\nWe tried to normalize the citH3 value to the first time point but found that this approach\nwould not reflect the differences between the treatment groups.\n3. Figure 6C: Interesting to note that no CitH3 was detected in cells treated with 3 ug of\nTAT-PYC-Smad7 while this amount of the biologics minimally impairs NETosis based on\nfigure 5.\nResponse: In Figure 6C, cell lysates were collected only at 30 minutes after PMA\ntreatment, whereas in Figure 5 we performed longitudinal observations. The inhibitory\neffect of low-dose Tat-PYC-Smad7 may become less pronounced over time compared to\nhigher doses. Nevertheless, as shown in Figure 5D, even the low-dose Tat-PYC-Smad7\ntreatment produced a statistically significant reduction in NETosis.\n4. Figure 6E: How long after PMA stimulation? does it match with a limited co-localization\nobserved in 6G?\nResponse: The data in Figure 6E (Sup. Fig. 15 in revised manuscript) were collected 1\nhour after PMA stimulation, consistent with the early stage of NETosis shown in the\nrevised Fig.6G. At this timepoint, we see limited NETosis but we do see MPO\ncolocalization with Tat-PYC-Smad7-HA in the nuclei of some cells. This biochemical\napproach supports an interaction between Smad7 and MPO, although the imaging results\nare more informative for where the interaction is occurring."}, {"round": 3, "reviewer_comments": {"reviewer_1": "Reviewer：The authors have commendably addressed all the raised concerns with substantial revisions. To enhance the robustness of the in vivo data, they have increased the sample size and re-evaluated the relevant parameters. Furthermore, they provided further validation of blood glucose levels in the knockout mice, effectively clarifying this issue. Building on this, they systematically verified the therapeutic effect of the target gene through both local injection and systemic administration in diabetic mice. Finally, to substantiate the involvement of NETosis, the authors incorporated new staining experiments and convincingly demonstrated its correlation with wound healing outcomes.", "reviewer_5": "Congratulations to the authors for the revisions. My major questions are answered and the manuscript should be published, in my opinion.", "reviewer_6": "This study is well-designed and highly significant, exploring a Smad7-based protein agent (Tat-PYC-Smad7) for diabetic wound healing. While the reviewers rightly noted that the mechanism linking Tat-PYC-Smad7 and MPO needs more work, we agree with the authors' statement that fully elucidating this may require a new, systematic study. For now, the authors have adequately addressed the points raised by Reviewer 2. The study doesn’t just confirm Smad7’s known role in blocking the TGFβ/NFκB pathway—it also shows that Smad7 directly inhibits neutrophil NETosis. However, neutrophils play a dual role in wound healing, being crucial for clearing pathogens. Although this research doesn’t cover infection, real-world diabetic wounds are frequently infected. We believe this is worth keeping in mind, as it could potentially influence the future clinical application of Tat-PYC-Smad7."}, "author_response": ""}]

Decision letter