bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 5600: GTP Cyclohydrolase/BH4 Pathway Protects EPC Function via Suppressing Oxidative Stress and Thrombospondin-1 in Salt-Sensitive Hypertension
Authors: Xie He-Hui , Channon Keith M , Chen Alex F
Published: April 2024

Background:
Endothelial progenitor cells (EPC) are both reduced and dysfunctional in hypertension that correlates inversely with its mortality, but the mechanisms are poorly understood. eNOS critically regulates EPC mobilization and function, but is uncoupled in DOCA-salt hypertension due to reduced essential cofactor tetrahydrobiopterin (BH4). We tested the hypothesis that GTP cyclohydrolase I (GTPCH), the rate-limiting enzyme of BH4 synthesis, protects EPC function in DOCA-salt mice.

Methods and Results:
EPCs were isolated from peripheral blood and bone marrow of wild-type (WT), WT DOCA-salt, endothelial-specific GTPCH transgenic (Tg-GCH), DOCA-salt treated Tg-GCH, and BH4 deficient hph-1 mice. Both EPC adhesion and angiogenic functions were impaired in WT DOCA (58 and 38%, respectively, n=5– 8, p<0.01) and hph-1 mice (40 and 31%, respectively, n=5–8, p<0.01, vs. WT), which were rescued in DOCA Tg-GCH mice (93 and 92%, respectively, n=6–7). Superoxide (O
2
−
, DHE-) and NO (DAF-FM flow cytometry) levels in EPCs were elevated and reduced, respectively, in WT DOCA (220% and 53%, respectively, n=5–12, p<0.01) and hph-1 mice (280% and 41%, respectively, n=6–12, p<0.05, vs. WT), which were blunted in DOCA Tg-GCH mice (137% and 96%, respectively, n=6–7). Thrombospondin-1 (TSP-1), a secreted potent anti-angiogenic protein, was elevated in EPCs of WT DOCA and hph-1 mice (290 and 280%, respectively, n=8–12, p<0.01 vs. WT), but not in DOCA Tg-GCH mice (160%, n=9–12, p>0.05). In vitro treatment of EPCs from WT DOCA mice with BH4, PEG-SOD, or L-NNA significantly reduced TSP-1 (51, 42 and 53%, respectively, n=5–12, p<0.01) and O
2
−
levels (58, 67 and 69%, respectively, n=5– 6, p<0.01 vs. WT-DOCA). NO level in EPCs of WT DOCA mice was rescued by BH4 and PEG-SOD (144 and 165%, respectively, n=4–7, p<0.05 vs. control), but not L-NNA. Crossing eNOS KO and Tg-GCH mice resulted in preserved circulating EPC number (88%, n=4–15, p>0.05 vs. WT), reduced EPC O
2
−
level (64%, n=4 – 8, p<0.05 vs. WT-DOCA), and improved EPC adhesion and angiogenic functions (160 and 158%, respectively, n=4 –12, p<0.05 vs. WT-DOCA).

Conclusion:
We demonstrate for the first time that GTPCH/BH4 pathway protects EPC function via suppressing oxidative stress and TSP-1 level in salt-sensitive hypertension.

This research has received full or partial funding support from the American Heart Association, Midwest Affiliate (Illinois, Indiana, Iowa, Kansas, Michigan, Minnesota, Missouri, Nebraska, North Dakota, South Dakota & Wisconsin).
Abstract 5611: ETS2 Contributes to a Pro-atherogenic Endothelial Cell Phenotype, Causing Plaque Destabilization in Advanced Atherosclerotic Lesions
Authors: Cheng Caroline , Tempel Dennie , den Dekker Wijnand , Haasdijk Remco , Herpers Robert , Bos Frank , van de Kamp Esther , Blonden Lau , Schulter-merker Stefan , Duckers Henricus J
Published: April 2024

Neovascularization is a requirement for embryonic development and plays an important role in the pathogenesis of various diseases in adults. In atherosclerosis, the role of neovascularization remains to be elucidated. Previously, we conducted a genome-wide microarray analysis for vasculogenesis during murine embryonic development. The
E
26
T
ransformation-specific
S
equence 2 (ETS2) transcription factor was upregulated in Flk1+ angioblasts and vascular expression of ETS2 was validated by whole mount
in situ
hybridization in the developing zebrafish. We sought to assess the role of ETS2 on neovascularization during atherosclerotic lesion progression toward plaque instability. We conducted experiments in ApoE−/− mice using a vulnerable plaque (VP) model that creates vulnerable and stable plaques in the common carotid artery by flow alteration. QPCR analysis showed that ETS2 expression is augmented in vulnerable versus stable lesions (P<0,05, N=3). Stimulation with the pro-atherogenic cytokine TNF
α
increased ETS2 expression and induced ETS2 translocation from the golgi area to the nucleus. Western Diet increased ETS2 expression on the endothelium of the aorta of ApoE−/− animals compared with chow-fed controls, indicated by whole mount staining (P<0,05, N=4).
In vitro
knockdown of ETS2 by SiRNA in HUVECs diminished the expression of the cytokines MCP1, IL6, and the adhesion molecule VCAM1, under basal conditions and in response to TNF
α
(P<0,05, N=4). ETS2 knockdown by SiRNA impeded new vessel formation in a matrigel tube-formation assay (P<0,05, N=8).
In vivo
, adenoviral transduction of ETS2 in the adventitia of the VP region significantly augmented the vascular network of the adventitia compared with shamvirus treated controls. ETS2 overexpression increased both lesion size and necrotic core area, and promoted plaque destabilization by increasing intraplaque lipid and macrophage accumulation, while vascular smooth muscle cell accumulation was diminished. In conclusion, ETS2 expression correlates with plaque instability and responds to pro-atherogenic stimuli. Subsequently, ETS2 function is crucial for induction of the pro-atherogenic EC phenotype and promotes destabilization in advanced atherosclerotic lesions.
Abstract 5612: Evidence for Posttranscriptional Control of Individual Members of the miR-17–92 Cluster
Authors: Bonauer Angelika , Fischer Ariane , Zeiher Andreas M , Dimmeler Stefanie
Published: April 2024

MicroRNAs (miRs) are small noncoding RNAs that regulate gene expression by binding to the cellular transcript leading to translational repression or degradation of the target mRNA. The miR-17–92 cluster encodes miR-17, miR-18a, miR-19a, miR-20a, miR-19b and miR-92a. Several members of the miR-17–92 cluster regulate angiogenesis and inhibition of miR-92a was recently shown to enhance recovery after ischemia in animal models. Because all members of the miR-17–92 cluster are under the control of the same promoter and are transcribed by one primary transcript, the mature miRs are expected to be regulated similarly. However, when we assessed the expression of the individual miRs in different cell types using a microRNA array, specific miRs were enriched. In HUVEC, miR-19b (51±3), miR-20a (13±1) and miR-92a (11±0.5) were highly expressed, whereas in CD34+ hematopoietic progenitor cells, miR-17 (27±3), miR-19b (88±13), miR-20a (94±19) were expressed at highest levels. MiR-18a expression was similar in both cell types (1.6±0.1 versus 1.8±0.1) indicating that specific members of the cluster are selectively processed or stabilized. To determine the regulation of the individual mature miRs in endothelial cells, HUVEC were incubated under hypoxia. Whereas the primary miR-17–92 cluster was not regulated, the mature forms of miR-19a (257+145%, 72h) and miR-92a (246±72%, 72h) were increased although with a different time course. Likewise, the individual members of the miR-17–92 cluster were significantly up-regulated after induction of hind limb ischemia (miR-92a: 4-fold; maximum at day 2) and miR-19a (6-fold; maximum at day 3), whereas miR-18a was not significantly regulated. In summary, these data provide first evidence for the posttranscriptional regulation of the miR-17–92 cluster, which controls neovascularization. Ongoing studies aim at identifying miR binding proteins in order to further characterize the mechanisms underlying the posttranscriptional regulation of the miR-17–92 cluster.
Abstract 5638: Autologous CD34+ Cell Therapy for Refractory Angina: 12 Month Results of the Phase II ACT34-CMI Study
Authors: Losordo Douglas W , Henry Tim , Schatz Richard A , Lee Joon Sup , Costa Marco , Bass Theodore , Schaer Gary , Niederman Alan , Mendelsohn Farrell , Davidson Charles , Waksman Ron , Soukas Peter A , Simon Daniel , Chronos Nicolas , Fortuin F David , Huang Paul P , Weintraub Neal , Yeung Alan , Rosenfield Kenneth , Wong S Chiu , Taussig Andrew , Rava Amish N , Sherman Warren , Kereiakes Dean , Strumpf Robert K , Port Steven , Pieper Karen , Adams Peter X , Harrington Robert
Published: April 2024

Background:
Refractory angina(Ang) effects 150 –250,000 pts in the US alone. These individuals have exhausted options for revasc and continue to have lifestyle limiting ang despite optimal medical Rx. Pre-clinical studies provided evidence that human CD34+ cells(CD34) can stimulate new blood vessel formation in ischemic myocardium(Myo), improving perfusion and function. We performed a phase 2 study of intraMyo injection of autologous CD34 in pts with CCS Class 3 and 4 Ang to obtain evidence for feasibility, safety and bioactivity.

Methods:
A phase 2 randomized, double-blind, placebo-controlled clinical trial was performed at 26 centers in the United States. There were 3 treatment groups: placebo, low dose (1×10^5 CD34/kg body wt) and high dose (5×10^5 CD34/kg). All pts underwent mobilization with GCSF 5 mcg/kg/day SC for 5 d, followed by apheresis on d5 to collect mononuclear cells. On day 6 CD34 were purified from the collected cells using the Isolex 300i device. After lot release testing, CD34 were injected endocardially at 10 locations in the ischemic Myo using the Myostar catheter following NOGA mapping. Placebo injections consisted of identical volumes of the diluent only.

Results:
A total of 167 pts were randomized and completed the injection procedure and 162 completed the 6 mo evaluation. The mean age of subjects was 61.0±8.9, there were 22 female and 145 male subjects. Trop was elevated in 26 pts at baseline and in 71 at some point following GCSF or injection. At 6 mo CD34 treated pts showed more reduction in ang frequency (−8.6 placebo vs 14.2 low dose) and improved ETT time (+69 sec placebo vs. +138 low dose). At 12m CD34 treated continue to show more reduction in angina (−8.2 placebo vs. −15.0 low dose) and improved ETT (+58 sec placebo vs. + 139 sec low dose). For key endpoints the high dose group results were inferior to low dose. The 12m data set has been locked and is being analyzed and the results of the completed analysis will be presented.

Conclusions:
Autologous CD34 cell therapy was associated with improved exercise tolerance and reduced angina in no-option pts with intractable angina. Ongoing analysis will determine endpoints, sample size and suitability of this therapy for a phase III study in no-option pts with refractory angina.
Abstract 5669: Monocytic Cells Are Required for Endothelial Regeneration
Authors: Werner Nikos , Haghighat Bita , Engel Daniel , Kurts Christian , Nickenig Georg , Wassmann Sven
Published: April 2024

Background: Circulating endothelial progenitor cells (EPC) contribute to endothelial cell (EC) regeneration and are predictors for cardiovascular events. Systemic treatment with EPC fosters the repair of damaged endothelium. We have shown that transfusion of mononuclear cells (MNC) lead to an even more pronounced regeneration of the endothelium compared to EPC transfusion. Here, we determine the role of monocytic CD11b+ cells in EC regeneration.

Methods/Results Re-endothelialization, neointima formation, and endothelial function was determined in wild-type (WT) mice treated with intravenous injections of spleen-derived MNC, CD11b-depleted MNC, CD11b+ cells, or cell-free vehicle after EC denudation. EC regeneration was enhanced and neointima formation reduced after transfusion of total MNC and CD11b+ cells but not after CD11b-depleted cells. We further determined the role of monocytes in a model of a disseminated EC damage. In ApoE−/− mice, transfusion of CD11b+ cells and total MNC but not CD11b-depleted MNCs improved endothelial function. To determine the role of endogenous monocytes on EC repair, mice were treated with clodronate-liposomes (10
μ
l/g bw) to deplete monocytes. Re-endothelialization after focal EC denudation was significantly diminished in clodronate-treated compared to vehicle-treated mice. Next, we selectively depleted CD11b+ monocytes and tissue-residual macrophages using CD11b-DTR transgenic mice. Treatment with diphtheria toxin (DT) results in the complete ablation of circulating monocytes/tissue-resident macrophages. DT treatment was performed for 10 days (15ng/g bw). At day 5 post EC damage, the denuded area was significantly larger in DT treated compared to vehicle treated mice. Transfusion of Sca1+ cells significantly enhanced re-endothelialization in vehicle-treated CD11b-DTR mice but not in DT treated mice. Finally, transfusion of CD11b+ cells in CD11b-DTR mice treated with either vehicle or DT resulted in a comparable enhancement of re-endothelialization.

Conclusion Here we demonstrate that circulating CD11b+ monocytes contribute to EC regeneration. Depletion of circulating monocytes is associated with a delay in re-endothelialization after EC damage and is not rescued by Sca1+ cells.
Abstract 5755: The Role of Adhesion and Migration of Stimulated Multipotent Stromal Cell With EGF in Vasculogenesis in Type 2 Diabetes
Authors: Amin Ali H , Elmageed Zakaria Y Abd , Partyka Megan , Ismail Mohammed F , Elazeik Osman Y , Belmadani Souad , Matrougui Khalid
Published: April 2024

Background: Chronic tissue ischemia due to defective vascular perfusion is a marker of peripheral artery disease for which minimal therapeutic options exist. Thus, type 2 diabetes is characterized with altered stem cells and vasculogenesis. Because of the capability of stem cells to differentiate into vascular cells and the importance of epidermal growth factor (EGF) in angiogenesis, their critical role in the treatment of type 2 diabetic patients with ischemia peripheral artery disease may be as prophylactic agents preventing tissue damage by stimulating vasculogenesis. Therefore we determined if multipotent stromal cells (MSC) tagged with enhanced green fluorescent protein (EGFP) stimulated with or without exogenous EGF would improve vasculogenesis in ischemia hind limb of type 2 diabetic (db−/db−) mice.

Methods and Results: Isolated bone marrow from db−/db− mice showed an increase in oxidative stress and reduction in Akt compared to control. Ischemia was induced in the hind limb of db−/db− mice and control for 28 days. EGFP-MSC stimulated with or without exogenous EGF (10 ng/ml) for 24 hr were locally injected in the mice hind limb once a week for 4 weeks. After 4 weeks, blood flow measured with MoorLDI-Laser and angiogenesis assessed with X-ray were completely recovered in control mice compared to db−/db− mice. Interestingly db−/db− mice display a significant improvement of blood flow (60% and 96%) and vasculogenesis (61% and 98%) when treated with MSC or MSC pre-stimulated with exogenous EGF, respectively. Western blot analysis on hind limb muscles revealed an increase in phosphorylated Akt and VEGF receptor in db−/db− mice injected with MSC or MSC+EGF compared to non-treated db−/db− mice. Fluorescent microscopy images indicate that EGFP-MSCs differentiate into new microvessels. In vitro studies showed that adhesion and migration of MSC on cultured microvascular endothelial cells were elevated when MSC were pre-stimulated with EGF compared to non-stimulated MSC.

Conclusion: Our novel data provide evidence that in type 2 diabetes, MSC stimulated with EGF are able to significantly improve, better than MSC alone, the formation of microvessels in response to ischemia.
Abstract 5762: A Randomized Double-Blind Study to Assess the Onset and Offset of the Antiplatelet Effects of Ticagrelor versus Clopidogrel in Patients With Stable Coronary Artery Disease
Authors: Gurbel Paul , Bliden Kevin , Butler Kathleen , Tantry Udaya , Teng Renli , Antonino Mark , Patil Shankar B , Karunakaran Arun , Kereiakes Dean , Paris Cordel , Rsch Dolby , Purdy Drew , Saucedo Jorge , Wilson Vance , Ledley Gary , Storey Robert F
Published: April 2024

Background:
Rapid and consistent inhibition of platelet aggregation (IPA) confers protection against the occurrence of ischemic events, and reversible inhibition may be desirable for patients who require surgery. The ONSET-OFFSET study was conducted in patients with stable coronary artery disease (CAD) to characterize the onset and offset of IPA associated with ticagrelor (T), the first reversibly binding oral P2Y
12
receptor antagonist, and compare them with those associated with clopidogrel (C).

Methods:
In a multicenter, randomized, double-blind, parallel-group study, 123 patients with stable CAD currently receiving aspirin (75–100mg) were randomized to doses of T (180 mg loading, 90 mg bid maintenance [n=57]), C (600 mg loading, 75 mg qd maintenance [n=54]) or placebo (n=12) for 6 weeks. Curves for T for onset of IPA (final extent, 20 uM ADP) post loading dose and offset of IPA were compared to those for C. Onset treatment effects were analyzed by the Wilcoxon rank sum test; offset treatment effects were analyzed by random coefficients model.

Results:
After the loading dose, more rapid onset of IPA occurred with T compared with C at 0.5 h (41±33% vs 8±10%, p<0.0001), 1 h (79±25% vs 23±26%, p<0.0001), and 2 h (88±15 % vs 38±32%, p<0.0001). Higher IPA for T was consistently maintained during maintenance therapy (p<0.0001 at all times). More patients in the T group than in the C group achieved >50% IPA (98% vs 31%, p<0.0001) and >70% IPA (90% vs 16%, p<0.0001) at 2 h post loading dose. At 24 h after the last dose of T, mean IPA was 58% for T vs 52% for C (p=NS). A faster offset rate for IPA was observed after the last dose of T than for C from 4 to 72 h [slope (% IPA/h): −1.037 vs. −0.482, p<0.0001]. Mean IPA for T at 72 h postdose (Day 3) was comparable to C at 120 h (Day 5). Mean IPA on Day 5 for T was similar to that for C on Day 7, both of which were not statistically different from placebo (p<0.05).

Conclusions:
Ticagrelor achieves more rapid and greater sustained IPA than clopidogrel. Moreover, after drug cessation the antiplatelet effect of ticagrelor had a faster offset. These results suggest value of ticagrelor when treatment protocol calls for rapid platelet inhibition or prompt offset of effect.
Abstract 5763: GP IIb/IIIa Inhibitors During Primary Percutaneous Coronary Intervention for STEMI: A Meta-Analysis of Randomized Controlled Trials
Authors: Tamhane Umesh U , Meier Pascal , Chetcuti Stanley , Grossman P. Michael , Gurm Hitinder S
Published: April 2024

Background: Earlier meta analyses demonstrated a survival benefit with adjunctive GP IIb/IIIa inhibitors (GPI) during primary percutaneous interventions (pPCI) for STEMI. Recent pPCI trials evaluating the role of GPI have demonstrated conflicting results. We performed a comprehensive meta-analysis to evaluate the role of adjuvant GPI therapy in patients undergoing pPCI.

Methods: Our meta-analysis included 15 randomized trials (n= 11407 patients) comparing adjunctive GPI therapy with heparin or bivalirudin in patients undergoing pPCI for STEMI. Summary odds ratios for safety and efficacy endpoints at 30 days were calculated using random-effects models.

Results: There was no significant difference in the incidence of 30-day mortality (Odds ratio [OR] 0.90, 95% CI 0.65–1.23, p = 0.49), reinfarction (OR 0.84, 95% CI 0.61–1.15, p = 0.27) or stroke (OR 0.68, 95% CI 0.35–1.30, p = 0.25). GPI therapy was associated with a significant reduction in rates of target vessel revascularization (OR 0.67, 95% CI 0.53 – 0.85, p = 0.001) and stent thrombosis (OR 0.51, 95% CI 0.32 – 0.83, p = 0.006). The risk of major bleeding (OR 1.72, 95% CI 1.39 –2.14, p< 0.001) and thrombocytopenia (OR 2.2, 95% CI 1.63–2.98, p< 0.001) was significantly increased in the GPI regimen.GPI therapy would prevent approximately 9 ST at the cost of 10 major bleeding episodes for every 1000 STEMI patients.

Conclusion: Adjuvant GPI therapy is not associated with improved survival compared to control therapy in STEMI patients undergoing pPCI. It significantly reduced ST and TVR at the cost of increase in major bleeding and thrombocytopenia.
Abstract 5769: Cardiovascular Outcomes for Patients Treated With Ezetimibe/Simvastatin Combination Compared With Simvastatin or Atorvastatin Alone
Authors: Stockl Karen M , Le Lisa , Zhang Shaoang , Harada Ann S , Solow Brian K , Ramsey Scott D
Published: April 2024

Purpose:
The efficacy of the combination of ezetimibe and simvastatin has been questioned following the results from the ENHANCE study, which found no difference in change in carotid intima-media thickness with ezetimibe/simvastatin combination compared with simvastatin alone. Because of the limited patient population (heterozygous familial hypercholesterolemia) and failure to examine cardiovascular events, additional research is necessary to evaluate clinical outcomes for patients using ezetimibe/simvastatin. Although a controlled clinical outcomes study is currently being conducted, results are not expected until 2012.

Methods:
Using administrative claims from a health insurance plan that included commercial and Medicare clients, we conducted a retrospective cohort analysis to compare the relative rates of clinical events (hospitalization for myocardial infarction (MI) or stroke) among patients age 18 to 84 with history of MI, stroke, or diabetes treated with ezetimibe/simvastatin combination versus simvastatin alone or atorvastatin alone. Simvastatin and atorvastatin-treated patients were each matched 1:1 with ezetimibe/simvastatin patients based on simvastatin daily dose equivalent and propensity score (derived using demographic and cardiovascular risk variables). Proportional hazards regression was performed with adjustment for demographics, medical history, and coronary heart disease medication use.

Results:
There were 9,983 patients in each cohort. The number of events per 100 person-years was 0.89 for ezetimibe/simvasatin, 0.93 for atorvastatin, and 1.05 for simvastatin. The risk of events was not significantly different for ezetimibe/simvastatin vs. atorvastatin alone (adjusted HR 0.89; 95% CI, 0.67–1.17), for ezetimibe/simvastatin vs. simvastatin alone (adjusted HR 0.80; 95% CI, 0.61 – 1.04), or simvastatin alone vs. atorvastatin alone (adjusted HR 1.11; 95% CI, 0.86 –1.44).

Conclusions:
Although the ENHANCE study raised questions on the efficacy of the ezetimibe/simvastatin combination, findings from this large population of high risk patients suggest that cardiovascular outcomes for patients treated with ezetimibe/simvastatin are similar to those treated with simvastatin or atorvastatin alone.
Abstract 5775: C57BL/6 NK Cell Gene Complex Crucially Involved in General Vascular Remodelling
Authors: Seghers Leonard , van Weel Vincent , van Bergen Jeroen , Toes Rene , Hellingman Alwine , de Vries Margreet , van Hinsbergh Victor , Quax Paul
Published: April 2024

Background
The immune system plays an important role in vascular remodelling. Inflammatory cells as monocytes, CD4+ and CD8+ T cells were already proven to be modulators of vascular remodelling. Recently, NK cells were indicated to contribute to vascular remodelling in processes of revascularisation and atherosclerotic lesion development. Interestingly, C57BL/6 mice display profound induced vascular remodelling, eg in arteriogenesis, restenosis and vein grafting, whereas BALB/c mice display much less remodelling. Moreover, these two mouse strains are different in their NK cell receptor gene complex (NKC), which codes for activating and inhibitory NK cell receptors.

Aim
To demonstrate that C57BL/6 NKC is crucially involved in modulating vascular remodelling in general.

Material & Methods
C57BL/6, BALB/c and BALB.B6-
CMV1

r

(CMV1
r
) mice, of which the latter is congenic for the C57BL/6 NKC, were used in two different models of vascular remodelling, induction of restenosis by femoral artery cuff placement and vein grafting by placement of a venous interposition in the mouse carotid artery. Vascular remodelling was monitored by morphometry and immunohistology.

Results
After cuff-placement the congenic CMV1r mice showed progressive formation of neointima (4745 um2), comparative to C57BL/6 (4459 um2), and significantly more than BALB/c mice (1247 um2), that displayed very little formation of neointima. Vein graft remodelling in CMV1
r
mice showed stronger intimal hyperplasia in vein grafts (0.22mm2) compared to both C57BL/6 mice (0.15mm2) and BALB/c mice (0.11mm2). Importantly, CMV1
r
displayed an inflammatory infiltrate similar to that of C57BL/6 mice, whereas BALB/c mice show hardly any inflammatory cell influx into the vessel wall.

Conclusion
These data demonstrate that the C57BL/6 NK cell gene locus is involved in the induction of profound vascular remodelling in general. Furthermore, the observed differences in inflammatory infiltrate between the three mouse strains strongly suggest that the C57BL/6 NK receptor repertoire might be involved in triggering an immune response that is associated with profound vascular remodelling.
Abstract 5791: A New Score to Improve the Detection of Peripheral Artery Disease
Authors: Duval Susan , Jaff Michael R , Boden William E , Alberts Mark J , Califf Robert M , Eagle Kim A , D’Agostino Ralph B , Massaro Joseph M , Pedley Alison , Steg P. Gabriel , Bhatt Deepak L , Hirsch Alan T
Published: April 2024

Background:
Detection of peripheral artery disease (PAD) typically entails noninvasive imaging or population screening, but whether a risk factor-based model has clinical utility is unclear. Our objective was to derive and internally validate a new score for estimating the probability of PAD utilizing the large US REACH Registry dataset.

Methods:
Analyses were conducted on 23,395 US REACH Registry outpatients with a complete baseline risk factor profile. PAD presence was determined by previous or current intermittent claudication associated with an ankle-brachial index <0.9 or previous lower extremity intervention. Multivariable stepwise logistic regression identified cross-sectional correlates of PAD from demographic, clinical, and laboratory variables. Model performance was assessed internally using 10-fold cross validation, and effect estimates were used to generate the PAD score.

Results:
Overall PAD prevalence was 9.1% (n=2135), with a mean age of 70 years. Age, smoking, history of congestive heart failure, diabetes, coronary artery disease (CAD), cerebrovascular disease (CVD), BMI, sex, and hypertension stage were predictive of PAD (Table 1

). The model had reasonable discrimination on derivation and internal validation (c-index = 0.61 and 0.60, respectively). The model-estimated PAD prevalence varied more than 3-fold from lowest to highest decile (range 4.8% to 16.4%) and corresponded closely with the actual PAD prevalence observed in each.

Conclusions:
This new PAD detection score uses easily available clinical variables to estimate the probability of disease prevalence. While the predictive power of the model may be limited, its use should nevertheless improve PAD detection in vulnerable, at-risk populations. These data should provide clinicians and patients with a simple risk-based tool to estimate the probability of PAD prevalence across a wide range of patients with atherothrombosis.

Table 1:

Factors related to PAD
Abstract 5801: Experimental Identification of Hypoxia-induced miR-210 Targets
Authors: Fasanaro Pasquale , Greco Simona , Banfi Cristina , Pescatori Mario , Ivan Mircea , Capogrossi Maurizio C , Martelli Fabio
Published: April 2024

MicroRNAs (miRNAs) are small non-protein-coding RNAs that are incorporated into the RNA-induced silencing complex (RISC) and inhibit gene expression by regulating the stability and/or the translational efficiency of target mRNAs. Previously, we demonstrated that miR-210 is a key player of endothelial cell (EC) response to hypoxia, modulating EC survival, migration and ability to form capillary like-structures. Moreover, the receptor tyrosine kinase ligand Ephrin-A3 was identified as one functionally relevant target. Since each miRNA regulates hundreds of mRNAs, different approaches were combined to identify new miR-210 targets.

Using target prediction softwares, 32 new miR-210 potential targets were identified.

The proteomic profiling of miR-210 over-expressing ECs identified 11 proteins that were specifically inhibited by miR-210, either directly or indirectly.

Affymetrix based gene expression profiles identified 51 genes that were both down-modulated by miR-210 and de-repressed when miR-210 was blocked.

Surprisingly, only few genes identified either by proteomics or transcriptomics were recognized as miR-210 targets by target prediction algorithms. However, a low-stringency pairing research revealed an enrichment for miR-210 putative binding sites, raising the possibility that these genes were targeted via non-canonical recognition sequences. To clarify this issue, miR-210-loaded RISC was purified by immuno-precipitation along with its mRNA targets. The presence of Ephrin-A3 mRNA in the complex validated this approach. We found that 32 potential targets were indeed enriched in miR-210-loaded RISC, and thus can be considered as genuine miR-210 targets. In keeping with these conclusion, we were able to further validate a sub-set of them by 3′UTR-reporter assays. Gene ontology analysis of the targets confirmed the known miR-210 activity in differentiation and cell cycle regulation, highlighting new functions such as involvement in RNA processing, DNA binding, development, membrane trafficking and aminoacid catabolism. In conclusion, we validated a multidisciplinary approach for miRNAs target identification and indicated novel molecular mechanisms underpinning miR-210 role in EC response to hypoxia.
Abstract 5809: Recombinant HSP27 Therapy Reduces Serum Cholesterol Levels and Experimental Atherogenesis
Authors: Chen Yong-Xiang , Zhao Xiaoling , McNulty Melissa , O’Brien Edward R
Published: April 2024

Introduction: Recently we demonstrated that Heat Shock Protein 27 (HSP27) is a biomarker of atherosclerosis and apoE
−/−
mice that over-express HSP27 show a marked attenuation of aortic lesion size compared to apoE
−/−
mice (ATVB 20005; Circ Res 2008). Moreover,

serum HSP27 levels are inversely related to atherosclerotic burden,

estrogens mediate the release of HSP27 from cells and

extracellular HSP27 binds to Scavenger Receptor-A where competitively inhibits acLDL uptake.

Purpose/Methods: The goal of this study was to determine if recombinant HSP27 (rHSP27) administered to apoE
−/−
mice can attenuate atherogenesis. rHSP27 was produced in E.coli; (endotoxin concentration <8EU/mg).
In vitro
aggregation and refolding assays revealed appropriate rHSP27 function. In vivo a series of subcutaneous dosing schedules were explored. Herein we report the results obtained for ApoE
−/−
mice maintained on a high fat diet and randomized to rHSP27 (100
μ
g) vs. phosphate buffered saline (PBS) twice a day for 3 weeks.

Results: All mice survived the study period without changes in body weight or adverse events. Total serum cholesterol levels were approximately 40% lower in rHSP27 treated mice. The aortic lesion areas were reduced by approximately 30% and 25% for
en face
and sinus cross-sectional analyses. In rHSP27 treated mice the abundance of aortic sinus lesion macrophages and apoptotic macrophages were reduced by approximately 45% and 80%, respectively.

Conclusions: rHSP27 is a promising new therapeutic modality for reducing serum cholesterol levels as well as atherogenesis. Moreover, the lesions that form in mice treated with rHSP27 contain fewer inflammatory cells and undergo less apoptosis. Studies are ongoing to optimize the dose, schedule and form of rHSP27 that might be used not only for the prevention of atherogenesis but also regression of existing lesions.
Abstract 5815: Therapeutic Targeting of the Calmodulin-Cyclin E Interaction in Vascular Smooth Muscle Cells
Authors: Hui Sonya , Choi Jaehyun , Zaidi Syed , Momen Abdul , Ban Kiwon , Husain Mansoor
Published: April 2024

Background:
Cell cycle regulation in vascular smooth muscle cells (VSMC) is a putative target in proliferative vascular diseases. Having shown that increases in intracellular [Ca
2+
] are tightly coordinated and critical for VSMC proliferation, we also discovered that binding of Ca
2+
/calmodulin (CaM) to cyclin E/cdk2 is necessary for Ca
2+
-sensitive G
1
-to-S phase progression in VSMC. To test the importance of this, we developed a synthetic
C
aM-
b
inding sequence (CBS) that blocked CaM-cyclin E interactions, prevented activation of cdk2, and abrogated Ca
2+
-sensitive G1-to-S transitions in VSMC. Of note, binding of CaM to another target protein, calcineurin, was not altered by CBS.

Objective:
To evaluate the physiological relevance of targeting CaM-cyclin E interactions in human VSMC in vitro and in a mouse model of neointima formation in vivo.

Methods & Results:
Human aortic SMC (HA-SMC) were treated with the CBS peptide conjugated to the viral TAT transduction domain to increase bioavailability. Peptide delivery of TAT-CBS was confirmed by immunochemistry. TAT-CBS caused a dose-dependent anti-proliferative effect in HA-SMC, as well as in human aortic endothelial cells and mouse embryonic fibroblasts (MEF) as measured by
3
H-thymidine incorporation. Importantly, these effects were lost in MEF from cyclin E1/E2 double knockout mice, further supporting target specificity for CaM/cyclin E. Moreover, TAT-CBS treatment was not cytotoxic, as it did not increase either LDH or caspase-3 release compared to controls. In vivo application of TAT-CBS in pluronic gel to the outside of injured mouse carotid arteries significantly decreased neointima formation and medial thickening without affecting re-endothelialization as compared to animals treated with vehicle-only or negative-control peptide.

Summary:
Inhibition of CaM binding to cyclin E/cdk2 with the TAT-CBS peptide significantly decreases HA-SMC proliferation in a dose-dependent manner in vitro. Moreover, application of TAT-CBS-containing gel to injured carotid arteries inhibits VSMC proliferation without affecting re-endothelialization. These data support

TAT-CBS as a novel therapy for restenosis, and

the general approach of targeting Ca
2+
-sensitive cell cycle control in VSMC.
Abstract 5820: Impact of Renal Function on Coronary Plaque Composition at Angiographically Severe Lesions
Authors: Miyagi Motohiro , Ishii Hideki , Murakami Ryuichiro , Isobe Satoshi , Hayashi Mutsuharu , Arai Kosuke , Matsubara Tatsuaki , Murohara Toyoaki
Published: April 2024

Background:
Recent studies have demonstrated that patients with chronic kidney disease are at high risk of arteriosclerosis. Recently, coronary plaque components can be evaluated by integrated-backscatter intravascular ultrasound (IB-IVUS) and lipid-rich plaque is associated with vulnerable plaque.

Objective:
The aim of this study was to investigate the relationship between renal function and tissue characterization of coronary plaque at the target stenotic site for percutaneous coronary intervention (PCI).

Methods:
We prospectively performed IB-IVUS before elective PCI to 89 consecutive patients with stable angina. According to estimated glomerular filtration rate (eGFR), they were divided into the two groups (eGFR < 60 ml/min/1.73m
2
or eGFR ≥ 60 ml/min/1.73m
2
). The tissue characteristics of the coronary plaque at each target stenotic site were evaluated by three-dimensional IB-IVUS just before PCI procedure.

Results:
Patients with eGFR < 60 ml/min/1.73m
2
had greater lipid volume and smaller fibrous volume compared to patients with eGFR ≥ 60 ml/min/1.73m
2
on the 3-D IB-IVUS image (36.7±10.6 % vs. 28.7±9.3 %, p < 0.001 and 59.1±8.7 % vs. 66.3±8.3 %, p < 0.001, respectively). eGFR showed a significant negative correlation with lipid volume and had a significant positive correlation with fibrous volume in coronary plaques (r = −0.44, p < 0.001, and r = 0.46, p <0.001, respectively).

Conclusion:
These data suggest that there is a progressive increase in lipid-rich plaque as renal function deteriorated. Our findings may explain the increasing risk of cardiovascular events in patients with renal dysfunction.
Abstract 16678: Multigenic Etiology of CHD With Heterotaxy Involving PCD and Cilia Genes
Authors: Li You , Yagi Hisato , Onuoha Ewenza O , Barmada Michael , Tsang Michael , Lo Cecilia
Published: April 2024

Introduction:
We recently showed patients with CHD associated with heterotaxy can exhibit respiratory ciliary dysfunction (CD) similar to that of patients with primary ciliary dyskinesia (PCD) - a ciliopathy with airway mucociliary clearance defects. As both PCD and heterotaxy have a 1 in 10,000 incidence, the finding of CD in heterotaxy patients likely reflects the dual role of motile cilia in airway clearance and left-right patterning. Thus, we hypothesize heterotaxy may arise from mutations in PCD genes and other cilia related ciliome genes.

Methods and Results:
CHD patients with heterotaxy, 13 with CD and 13 without CD (no-CD), were sequence captured followed by SOLiD sequencing at 40X coverage to identify sequence variants in all 13 PCD genes and nearly 1000 other ciliome genes. Coding variants were filtered against dbSNP and the 1000 Genomes databases, and novel coding variants (NCV) were validated by Sanger sequencing. Analysis of the 13 PCD genes revealed 10 NCV in 7 CD patients, and 5 NCV in 3 no-CD patients. The 3 PCD patients had 5 NCV, with 2 siblings having homozygous CCDC39 mutation (1072delA) known to cause PCD. Interestingly, CD-heterotaxy patient 9002 was heterozygous for the most common pathogenic
DNAI1
mutation, IVS1+2_3insT, a disease NCV not seen in the general population. As PCD is a recessive disorder, we evaluated NCVs found in
DNAH6
, a motor dynein gene not yet demonstrated to cause PCD.
DNAH6
NCVs were found in 9002 and one other CD and one PCD patient, but not in any no-CD patient.
Dnah6
morpholino (MO) knockdown (KD) in zebrafish embryos showed laterality defects and phenotypes consistent with heterotaxy, suggesting a role in CD and heterotaxy.

Conclusion:
Our study showed heterotaxy patients with CD can have heterozygous NCVs in PCD genes, including known disease causing
DNAI1
mutation. Functional analysis with zebrafish MO showed
DNAH6
KD can cause heterotaxy. We propose
DNAH6/DNAI1
double heterozygous mutation may contribute to CD and heterotaxy.
Abstract 16725: Transcatheter Aortic Valve Implantation in Patients With Severe Aortic Stenosis and Small Aortic Annulus: Propensity-Matched Comparison to Standard Aortic Valve Replacement
Authors: Dumont Eric , Doyle Daniel , Bagur Rodrigo , DeLaRochelliere Robert , Kalavrouziotis Dimitri , Pibarot Philipppe , Rodes-Cabau Josep
Published: April 2024

Background:
Few data exist on the results of transcatheter aortic valve implantation(TAVI) in patients with a small aortic annulus(AA) compared to standard aortic valve replacement(SAVR). We sought to examine valve hemodynamics and clinical outcomes of patients with a small AA who underwent TAVI versus SAVR.

Methods:
Between 2007 and 2011, 46 patients (mean age 79.1±9.4 years) with severe aortic stenosis and an AA diameter <19.5 mm (mean 18.3±0.85 mm) underwent TAVI with a 23-mm Edwards SAPIEN bioprosthesis. These patients were compared to 217 propensity-matched patients who underwent SAVR with a 19-mm valve (78% bioprosthesis). Echocardiographic parameters and clinical outcomes were assessed prior to discharge and at follow-up.

Results:
There was a greater incidence of postoperative atrial fibrillation (34.6% vs 15.2%, p=0.02), acute kidney injury (12.4% vs 8.7%, p=0.05), and blood product transfusion (83% vs 46%, p<0.001) in the SAVR group compared to the TAVI group. In-hospital mortality was 2.2% in the TAVI group and 6.5% in the SAVR group (p=0.06). One-year survival rate was similar between groups (TAVI 91.3% vs SAVR 87.1%, p=0.20). At hospital discharge, mean transaortic gradients were greater in the SAVR group (18.3 ± 7.2 vs 14.9 ± 9.3 mmHg, p=0.006). Mean indexed effective orifice area (IEOA) was lower in the SAVR group (0.66 ± 0.16 vs 0.84 ± 0.2 cm2/m2, p<0.001), and severe prosthesis-patient mismatch (IEOA < 0.65 cm2/m2) occurred more frequently in the SAVR group (53.3%) than in the TAVI group (9.3%) (p<0.001). Aortic regurgitation(AR) occurred in 72% of the patients in the TAVI group (trivial or mild: 70%, moderate: 2%) compared to 28% of the patients in the SAVR group (trivial or mild in all cases), p<0.001. Echocardiographic results remained unchanged at 1-year follow-up.

Conclusion:
In patients with severe AS and a small AA, TAVI is associated with lower postoperative morbidity and better valve hemodynamics but a higher incidence of trivial/mild residual AR as compared to SAVR. These results suggest that TAVI is a reasonable alternative to SAVR in elderly patients with small AA.
Abstract 16727: A First Description of Increased Glutaminolysis in Experimental Right Ventricular Hypertrophy; Association With Ischemia-Induced Activation of cMyc
Authors: Piao Lin , Fang Yong-Hu , Parikh Kishan , Toth Peter T , Marsboom Glenn , Archer Stephen L
Published: April 2024

INTRODUCTION
We have previously showed that in right ventricular hypertrophy (RVH) and RV failure there is a maladaptive metabolic switch from glucose oxidation to glycolysis. A similar metabolic shift occurs in many cancers. Another metabolic derangement in cancer that is a metabolic shift to use glutamine as a substrate, glutaminolysis, has not been studied in RVH. cMyc, an oncogenic transcription factor, cooperating with hypoxia-induced factor 1, induces both glycolysis and glutaminolysis in cancer cells. We hypothesized that cMyc activation induces glutaminolysis in RVH.

METHODS AND RESULTS
3 groups of Sprague-Dawley rats were studied: 1) Control; 2) Pulmonary artery banding (PAB), in which RVH is induced by a 1.3mm pulmonary artery band; 3) Monocrotaline-induced RVH, elicited by subcutaneous injection of monocrotaline (60mg/Kg). RVH occurred with PAB and monocrotaline, evident from increased RV/LV+septum (
P
<0.001 vs control). In an RV Langendorff model, total coronary flow collected from coronary sinus was reduced in PAB and monocrotaline models vs control (
P
<0.01). Staining for CD31 and lectin (both markers of endothelial cells) demonstrated that capillary density was reduced in both RVH models vs control (
P
<0.05). The RV in monocrotaline model had lower capillary densities than RV in PAB model (
P
<0.05). cMyc expression was significantly increased in RV in both RVH models (
P
<0.05 vs control). cMyc also was increased in LV in monocrotaline model (
P
<0.05 vs control) but was unchanged in LV in PAB model. mRNA expression of the glutamine transporters, SLC7A5 and SLC1A5, were increased in both RV and LV in monocrotaline RVH (
P
<0.05 vs control); whereas only SLC7A5 expression was elevated in PAB RVs (
P
<0.05 vs control).

CONCLUSION
There is an increase of glutaminolysis in RV in RVH models. This is associated with RV ischemia and capillary rarefaction. It is unclear whether the observed cMyc activation is caused by or induces the capillary rarefaction. However, it is likely cMyc accounts for the increased glutaminolysis which is a newly recognized metabolic abnormality in RVH models. Our study suggests the potential of therapeutic inhibition of glutaminolysis as a treatment for RVH and RV failure.
Abstract 16803: Endothelial Cells Communicate with Vascular Smooth Muscle Cells Via Transfer of microRNA Contained in Secreted Exosomes
Authors: Imbrie Gregory A , Zhao Jin , Lu Qing , Bauer Wendy , Karas Richard H
Published: April 2024

Background:
Endothelial cell (EC) mediated inhibition of vascular smooth muscle cell (VSMC) proliferation plays an important role in maintaining vascular wall homeostasis and preventing atherosclerosis. The mechanism that mediates this effect has not been clarified. MicroRNAs (miR) are ∼23nt RNAs that regulate many important cellular processes by post-transcriptional regulation of gene expression. Exosomes are small (30-100nm) vesicles secreted from cells, and previously shown to contain miR. We hypothesized that intercellular transfer of miRNA from EC to VSMC via exosomes contributes to the inhibition of VSMC proliferation.

Results:
We show that EC secreted exosomes contain abundant miRs and protect their contents from degradation by circulating RNases. The miR population in secreted exosomes differs significantly from the cytoplasmic miR population, indicating regulation of miR packaging in ECs. Conditioned media of ECs inhibited Human Aortic VSMC (HAVSMC) proliferation by 37.4% (n=4, p=0.0006) at 120 hrs, while exosome-depleted conditioned media loses this inhibitory effect (n=4, p=0.0001). Purified exosomes applied directly to VSMCs inhibited HAVSMC proliferation by 28.8% at 72hrs (n= 8, p= 0.0107) and by 26.9% at 96 hrs (n=8, p=0.0596). Similar results were found with Coronary Artery SMC proliferation (data not shown). EC-derived exosomes are taken up into recipient SMCs, as seen by ICC to an exosome marker, GFP-CD63, and by qPCR to transferred miRNAs. Transfection into VSMC of RNA purified from EC secreted exosomes is sufficient to inhibit SMC proliferation (34.8%, n= 9, p=0.0001), and provides a similar level of inhibition compared to whole exosome treatment (33.5%, n=9, p=0.001). The miRNA population of exosomes can be engineered through transfection of the progenitor EC; transfection with pre-mir-203 leads to a 317-fold increase in exosomal mir-203 levels, and a 126-fold increase in SMCs treated with EC-derived conditioned media.

Conclusions:
This is the first evidence that ECs can communicate with VSMCs via transfer of exosomal miRs and further, identify this process as a mechanism for EC-mediated inhibition of VSMC proliferation. These findings also identify exosomal miRs as a potential therapeutic agent in vascular disease.
Abstract 16814: A miRNA Signature of the Failing Right Ventricle
Authors: Paulin Roxane , Sutendra Gopinath , Dromparis Peter , Bonnet Sebastien , Michelakis Evangelos
Published: April 2024

INTRODUCTION:
Right Ventricular Failure (RVF) is the most important prognostic structure for both morbidity and mortality in pulmonary hypertension (PHT) but remains understudied compared to the left ventricle (LV). Many differences (including embryologic origin) separate the 2 ventricles and the transition from a compensated to decompensated stage occurs much earlier in the RV vs the LV, in response to pressure overload. MicroRNAs (miRNAs) have emerged as important determinants of LV development and failure, but nothing is known about RV miRNAs. We hypothesized that specific miRNAs signatures may characterize the transition from the fetal to adult and from a compensated (CRV) to a decompensated (DRV) RV.

METHODS/RESULTS:
We studied free RV wall tissue from neonatal (Neo) and rats with normal RV function (NRV, RVSP=38±2 mmHg, CO=102±2 ml/min and RV/LV+Septum=22±1), CRV (RVSP=66±8 mmHg, CO=80±13 ml/min, RV/LV+Septum=48±3, 2-3 weeks post-monocrotaline) and DRV (RVSP=52±2 mmHg, CO=67±10 ml/min, RV/LV+Septum=58±2, ascites, weight loss, 4-6 weeks post monocrotaline) (n=6 in each group). We used Taqman Low Density Arrays to study the expression of 377 miRNAs. Neonates were separated clearly from other samples in both principal component analysis and hierarchical clustering analysis. 14 miRNAs were identified as differentially expressed between CRV, DRV and NRV (ANOVA applied on fold-change obtained after standard normalization procedures were performed (U6, median, quantile)). Among them, 7 showed a different pattern of expression compared to published data on LV hypertrophy: miR-200b, -132, -338-3p, -328, -155, -92a and -208. miR-208 has received attention as critical regulator of LVH. We showed that 208 decreased in CRV and DRV compared to NRV, whereas its expression is not known to decrease in LVH. This was confirmed by individual qRT-PCR and miR-208 levels were also found to negatively correlate with RV size (r=-0.72, n=20, p<0.001) as failure progresses.

CONCLUSION:
Further exploration of this unique miRNA signature, compared to LV, may also open new avenues of investigation on RVF. Since miR-208 is cardiac specific and regulated differently compared to LVH, it may offer important clues on why the RV responds suboptimally to pressure overload.