Shaver et al. BMC Molecular and Cell Biology
https://doi.org/10.1186/s12860-024-00504-w

BMC Molecular andCell Biology(2024) 25:8

Open Access

RESEARCH

Mechanical stretch leads to increased
caveolin‑1 content and mineralization potential
in extracellular vesicles from vascular smoothmuscle cells
Mohammad Shaver1, Kassandra Gomez1, Katherine Kaiser1 and Joshua D. Hutcheson1,2*

Abstract
Background Hypertension-induced mechanical stress on vascular smooth muscle cells (VSMCs) is a known risk
factor for vascular remodeling, including vascular calcification. Caveolin-1 (Cav-1), an integral structural componentof plasma membrane invaginations, is a mechanosensitive protein that is required for the formation of calcifying
extracellular vesicles (EVs). However, the role of mechanics in Cav-1-induced EV formation from VSMCs has not beenreported.
Results Exposure of VSMCs to 10% mechanical stretch (0.5 Hz) for 72 h resulted in Cav-1 translocation into noncaveolar regions of the plasma membrane and subsequent redistribution of Cav-1 from the VSMCs into EVs. Inhibition of Rho-A kinase (ROCK) in mechanically-stimulated VSMCs exacerbated the liberation of Cav-1 positive EVs
from the cells, suggesting a potential involvement of actin stress fibers in this process. The mineralization potentialof EVs was measured by incubating the EVs in a high phosphate solution and measuring light scattered by the minerals at 340 nm. EVs released from stretched VSMCs showed higher mineralization potential than the EVs released
from non-stretched VSMCs. Culturing VSMCs in pro-calcific media and exposure to mechanical stretch increased
tissue non-specific alkaline phosphatase (ALP), an important enzyme in vascular calcification, activity in EVs released
from the cells, with cyclic stretch further elevating EV ALP activity compared to non-stretched cells.
Conclusion Our data demonstrate that mechanical stretch alters Cav-1 trafficking and EV release, and the released
EVs have elevated mineralization potential.
Keywords Mechanical stretch, Extracellular vesicles, Caveolin-1, Calcification, Vascular smooth muscle cells*Correspondence:
Joshua D. Hutcheson
jhutches@fiu.edu
1
Department of Biomedical Engineering, Florida International University,
10555 West Flagler Street, Engineering Center 2600, Miami, FL 33174, USA
2
Biomolecular Sciences Institute, Florida International University, Miami,
FL 33199, USA

Introduction
Vascular calcification results from bone-like mineral formation in the arterial wall and correlates significantlywith cardiovascular morbidity and mortality [1]. For decades, calcification was considered a degenerative process
due to aging; however, growing evidence has shown thatcalcification occurs in an actively regulated process similar to bone remodeling [2]. Calcification often begins in
specialized extracellular vesicles (EVs) released from vascular smooth muscle cells (VSMCs) in response to pathological stimuli [3]. Once released, these EVs sequester© The Author(s) 2024. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which
permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to theoriginal author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images orother third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line
to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory
regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of thislicence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Shaver et al. BMC Molecular and Cell Biology(2024) 25:8

calcium and phosphate ions to nucleate mineral formation [4]. Aggregation of the EVs leads to mineral growth,which disrupts the biomechanical function of the arterial wall and can directly contribute to both acute andchronic cardiovascular pathologies [5, 6].
Goettsch et al. reported that the formation of calcifying EVs requires the presence of caveolin-1 (Cav-1), a
structural protein important in the formation of plasma
membrane invaginations known as caveolae [7]. These
50–100 nm flask shape invaginations in the plasma
membrane contain abundant cholesterol, sphingolipids, and scaffolding proteins [8–11]. Caveolin-1 is the
major membrane protein in the caveolae structure andis involved in endocytosis, lipid trafficking, cell signaling, EV biogenesis, and mechanotransduction [12, 13].
Caveolae, in association with actin filaments, respondto mechanical stimulation such as membrane stretch[12, 14]. Increased tension on the cell membrane leads
to caveolae destabilization and disassembly [15]. Plasma
membrane deformation is associated with rapid cytoskeletal reorganization, which leads to caveolae endocytosis[8]. Caveolin-1 internalization and trafficking throughthe Golgi apparatus, begins the formation of calcifying
EVs [7, 16]. However, no studies have been conducted toinvestigate the role of mechanics in the generation of calcifying EVs.
Hypertension, a major risk factor for cardiovasculardiseases, abnormally increases mechanical stretch on thearterial wall [17, 18]. The main responders to mechanical stretch are VSMCs, which are abundant in the arterial
wall and responsible for the maintenance of acute vascular homeostasis through active contraction and relaxation [19–21]. Sustained pathological stressors cause
VSMCs to adopt a synthetic phenotype associated withincreased expression of osteogenic proteins and deposition of mineralized bone-like matrix [22]. Calcification
leads to arterial remodeling and alters arterial mechanics
in a way that reduces elasticity and increases the stiffness[23]. In vitro studies have modeled vascular calcificationby either culturing VSMCs in media designed to promoteosteogenic alterations in phenotype [24] or in mineralization-competent media that alter phosphate homeostasis[23]. Studies into the effects of mechanical stimulation
on caveolin-1 trafficking and EV formation could yield
new insight into how altered mechanics affects vascularremodeling and calcification in response to these pathological cues.
In order to examine the interrelation between mechanical stimulation, caveolin-1-dependent EVs formation,and the propensity of these vesicles to induce calcification, VSMCs were cultured in a cyclic strain environment
in vitro. First, we show that mechanical stretch redistributes caveolin-1 from VSMCs into EVs. Measurements ofPage 2 of 12

calcification potential of the released EVs demonstrated
enhanced mineralization competency in EVs fromstretched VSMCs.

Results
Mechanical stretch induces caveolin‑1 translocation
within plasma membrane of VSMCs

Due to their unique lipid and protein composition, caveolae exhibit a low buoyant density enabling separationfrom other cellular fractions through gradient centrifugation [25]. To investigate whether mechanical stretch
alters the subcellular localization of Cav-1 within VSMC
plasma membranes, caveolar and non-caveolar membrane fractions were separated by discontinuous sucrose
gradient ultracentrifugation. Immunoblotting analysis
revealed that in non-stretched control VSMCs, Cav-1
was detected in fractions 1–5. Following 72 h of cyclic
stretch, Cav-1 displayed a clear shift in localization tomore dense membrane fractions (Fig. 1). These data demonstrate that prolonged mechanical stretch induces thetranslocation of Cav-1 from caveolae domains to noncaveolar regions of the plasma membrane in VSMCs.
Mechanical stretch redistributes caveolin‑1 from VSMCs
into EVs

Next, we investigated the role of 72 h of cyclic mechanicalstimulation (0.5 Hz) in Cav-1 trafficking and EV releasein VSMCs. Quantitative analysis of Western blot data
demonstrated a statistically significant 1.39-fold reduction (P < 0.01) in cellular Cav-1 protein levels in mechanically stimulated VSMCs compared to static control cells(Fig. 2A). In contrast, Cav-1 was increased by 3.41-fold(P < 0.0001) in EVs secreted from VSMCs exposed to 10%
cyclic stretch versus EVs from non-stretched controls(Fig. 2B). Normalizing the Cav-1 content in EVs to theintracellular levels in corresponding VSMCs revealed a4.77-fold (P < 0.0001) higher EV-to-VSMC Cav-1 ratio in
the 10% cyclic stretch condition compared to the static
condition (Fig. 2C). Quantification of total intracellular cholesterol levels revealed that application of cyclicmechanical stretch for 24 h elicited a significant 1.96fold (P < 0.001) increase in cholesterol abundance within
VSMCs compared to non-stretch control cells (Fig. 2D).
Mechanical stretch leads to caveolin‑1 internalization

Immunofluorescence imaging revealed changes in Cav-1
localization within VSMCs after application of mechanical stretch. In non-stretched VSMCs, Cav-1 predominantly aligned along actin filaments (Fig. 3A), while
application of 10% equibiaxial cyclic stretch for 72 h
induced a redistribution of Cav-1 from actin-associatedregions to more dispersed regions within the cytosol(Fig. 3B). To investigate if Cav-1 mobilization in response

Shaver et al. BMC Molecular and Cell Biology(2024) 25:8

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Fig. 1 Effect of mechanical strain on subcellular localization of caveolin-1 in vascular smooth muscle cells (VSMCs). A Representative western blots
showing caveolin-1 (Cav-1) protein levels in caveolar (fraction 1) and non-caveolar (fraction 7) membrane fractions isolated from non-stretched
VSMCs and VSMCs exposed to 10% cyclic stretch for 72 h. B Densitometry analysis of Cav-1 revealed that cyclic stretch induced a significant
translocation of Cav-1 from the caveolar fraction to the non-caveolar fraction compared to non-stretched control cells n = 3 (*P < 0.05, ***P < 0.001).
Data represents mean ± SD. Full-length blots are presented in Supplementary Fig. 1

Fig. 2 Effect of cyclic strain on caveolin-1 distribution in vascular smooth muscle cells (VSMCs). A Application of 10% cyclic strain for 72 hsignificantly decreases the intracellular level of caveolin-1 compared to non-stretched control VSMCs, as quantified by Western blotting. B Exposureof VSMCs to 10% cyclic strain for 72 h significantly increases the release of caveolin-1 positive extracellular vesicles (EVs) compared to non-stretched
controls, as determined by Western blotting. C Cyclic strain results in increased trafficking of caveolin-1 from VSMCs into extracellular vesicles
as compared to static control conditions, based on normalization of extracellular vesicular caveolin-1 content to intracellular caveolin-1 levels
in corresponding VSMCs. D Cyclic mechanical strain elevates intracellular cholesterol levels in VSMCs. n = 9 Data are presented as mean ± SD(*P < 0.05, ****P < 0.0001). Data represents mean ± SD. Full-length blots are presented in Supplementary Fig. 2

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Fig. 3 Immunofluorescence staining shows Cav-1 (red) transitioning from alignment along actin filaments in non-stretched VSMCs)(A) to a moredispersed, cytosolic distribution after application of 10% cyclic strain for 72 h (B). C Pre-treatment with the dynamin inhibitor dynasore (40 μM)
reduces Cav-1 internalization in VSMCs subjected to 10% strain for 72 h compared to control. D This inhibition of Cav-1 endocytosis by dynasore
decreases Cav-1 release in EVs from stretched VSMCs by 2.41-fold (P < 0.01). E Levels of the exosomal marker CD63 are unchanged in EVs
from control versus 10% stretched VSMCs. F Knockdown of the Cav-1 gene in cyclically stretched VSMCs significantly decreased Cav-1 protein levels
in EVs released from these cells. n = 3. Data are mean ± SD (**P < 0.01) (Scale bar: 2 µm). Data represents mean ± SD. Full-length blots are presented
in Supplementary Fig. 3

to mechanical factors involved dynamin-dependentendocytosis, VSMCs were treated with the dynamininhibitor dynasore prior to 10% stretch for 72 h. Inhibition of dynamin activity resulted in the retention of Cav-1
along actin filaments (Fig. 3C), similar to the patternobserved in static VSMCs. This reduction in Cav-1 internalization upon dynasore treatment was accompanied
by a 3.41-fold (P < 0.01) decrease in the levels of Cav-1
released extracellular vesicles (Fig. 3D). Moreover, we did
not observe any significant change in the value of exosome marker CD63 in the isolated EVs of 10% stretched
VSMCs compared to non-stretched cells after 72 h
(Fig. 3E). Quantitative immunoblotting revealed a statistically significant 2.21-fold (P < 0.01) reduction in Cav-1

protein levels in EVs from stretched VSMCs transfectedwith Cav-1 siRNA versus scramble siRNA (Fig. 3F).
Actin filament dynamics regulate caveolin‑1 trafficking

Exposure of VSMCs to 10% mechanical stretch led toactin stress fiber reorientation in a uniform direction
compared to non-stretched cells (Fig. 4A and B) as indicated by phalloidin stain. Recent reports have described
the critical role of actin–myosin network in mobilizationand translocation of vesicles to the plasma membrane[26]. To investigate the role of actin filament dynamics
in stretch-induced Cav-1 trafficking, we inhibited RhoA
kinase (ROCK) activity using the small molecule Y-27632
to disrupt actin filament polymerization and contractility

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Fig. 4 RhoA kinase (ROCK) inhibition increases Cav-1 EVs independently of intracellular Cav-1 levels. Phalloidin staining shows reorientationof F-actin (green) in VSMCs subjected to 10% cyclic strain (A) versus non-stretched VSMCs (B). ROCK inhibitor Y-27632 (10 μM) disrupts F-actin
in both stretched (C) and static (D) VSMCs. This associates with increased EV Cav-1 release from stretched (E) and non-stretched (F) VSMCs treatedwith Y-27632 versus vehicle control. Intracellular Cav-1 levels are unaltered by ROCK inhibition in stretched (G) or static (H) VSMCs. n = 4. Data aremean ± SD. (*P < 0.05). (Scale bar: 10 µm). Data represents mean ± SD. Full-length blots are presented in Supplementary Fig. 4(Fig. 4C and D). Interestingly, ROCK inhibition significantly exacerbated Cav-1 EV content in both stretchedand non-stretched VSMCs (P < 0.05) (Fig. 4E and F).
ROCK inhibition did not change the intracellular level of
Cav-1 in either group (Fig. 4G and H).
Mechanical stretch induces mineral potential of liberated
EVs

To determine whether mechanical stretch changes thecalcifying potential of released EVs, we examined theconditioned media of VSMCs using a mineral formation assay. EVs from VSMCs exposed to 10% mechanical stretch exhibited a 20.9 ± 0.7% increase (P < 0.01) inthe maximum absorbance of 340 nm light compared to
EVs from non-stretched VSMCs (Fig. 5A and B). A linear regression during the rapid phase of mineralization revealed that the absorbance increase occurred
fourfold faster in the stretched samples compared tonon-stretched samples (Fig. 5C). Annexin V is a calciumbinding membrane protein that helps mediate mineral

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Fig. 5 A, B Light absorbance at 340 nm reveals that EVs from 10% stretched VSMCs form more calcific mineral. C Normalization with respect
to control samples reveals higher mineralization rate for 10% stretch. D Annexin V, a calcium binding membrane protein, in EVs increased
in response to increases in mechanical stretch. Collagen hydrogel assay to visualize EV-induced mineralization for stretched cells (E) compared
to non-stretched control (F). G Mechanical stretch significantly elevated the number of the mineral nucleation sites. H) Total mineralized area
was increased by EVs from mechanically stretched VSMCs. n ≥ 4. Data are mean ± SD (*P < 0.05, **P < 0.01). (Scale bar: 10 µm). Full-length blot
is presented in Supplementary Fig. 5

formation [27]. We observed 3.4-fold (P < 0.05) increasein Annexin V content in the isolated EVs of 10% stretched
VSMCs compared to non-stretched cells (Fig. 5D).
In order to directly visualize the impacts of mechanical stimulation on EV-mediated mineralization, a 3D
collagen hydrogel system was utilized as an in vitro biomimetic calcification model [6]. Quantitative image
analysis of the fluorescent micrographs was then carried out using ImageJ software to determine the number
and total area of mineralization sites within the hydrogels (Fig. 5E and F). Segmentation and particle analysis
revealed a 1.96-fold increase (P < 0.01) in the numberof mineral nucleation sites formed by EVs derived frommechanically stretched cells compared to non-stretched
ones (Fig. 5G). Additionally, total area of the mineralization sites was elevated by 2.2-fold (P < 0.01) in hydrogels
treated with EVs from mechanically stimulated cells versus non-stretch condition (Fig. 5H).
Cyclic stretch amplifies the pro‑calcific VSMC phenotype
induced by osteogenic culture

Culturing VSMCs in osteogenic media containing dexamethasone, ascorbic acid, and β-glycerophosphate
induced a significant 2.85-fold increase (P < 0.01)
in Cav-1 content in EVs released from the cells versus EVs from VSMCs maintained in normal growth
media (Fig. 6A). This is indicative of increased secretion of Cav-1-positive EVs under osteogenic conditions as shown previously [7]. Application of 10%
cyclic mechanical stretch further augmented Cav-1-EV

release from VSMCs cultured in osteogenic media.
Quantitative immunoblotting revealed a significant 3.5fold increase (P < 0.01) in Cav-1 levels in EVs released
from stretched VSMCs grown in osteogenic versus
normal media (Fig. 6A). Culturing VSMCs in osteogenic media led to increased Annexin V content in EVs
released from the cells. Quantitative immunoblotting
revealed a significant 1.88-fold increase in Annexin V
levels in EVs released from cyclically stretched VSMCs
following culture in osteogenic media compared tonormal media. A 1.86-fold elevation in Annexin V was
also observed in EVs from static VSMCs grown in osteogenic versus normal conditions (Fig. 6B).
Culturing VSMCs in osteogenic media led to a 6.86fold (P < 0.001) and 4.44-fold (P < 0.001) increase in tissue
non-specific alkaline phosphatase (ALP) activity in the
EVs released from cyclically stretched and non-stretched
VSMCs, respectively, compared to cells grown in normal media. This demonstrates induction of a pro-calcificphenotype under osteogenic conditions [6]. Interestingly,
a significant 1.7-fold increase (P < 0.01) in EV ALP activity was also observed in EVs from VSMCs cultured innormal growth media and subjected to cyclic mechanical stretch (Fig. 6C). Within the cells, osteogenic media
elicited a significant 13.5-fold and 13.2-fold increase in
intracellular ALP activity (P < 0.001) compared to normal
media for stretched and non-stretched VSMCs, respectively. Application of cyclic mechanical stretch reduced
intracellular ALP levels by 1.54-fold and 1.58-fold in osteogenic and normal culture media, respectively (Fig. 6D).

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Fig. 6 Osteogenic factors and cyclic stretch induce pro-calcific VSMC phenotype and EV release. A Osteogenic media increased EV Cav-1 1.85-foldand stretch augmented Cav-1 2.5-fold. B Osteogenic conditions elevated EV annexin V 0.88-fold and 0.86-fold with stretch and non-stretch.
C EV alkaline phosphatase (ALP) rose 5.86-fold and 3.44-fold in osteogenic media with stretch and non-stretch. Stretch also increased EV ALP
0.7-fold in normal media. D Cellular ALP increased 12.5-fold and 12.2-fold with osteogenic media and stretch reduced intracellular ALP 0.35-fold
and 0.36-fold. n = 6. Data are mean ± SD (**P < 0.01, ***P < 0.001, ****P < 0.0001). Full-length blots are presented in Supplementary Fig. 6

Discussion
The critical impact of hypertension-induced stress on
VSMCs and arterial remodeling—including calcification—has been well described in previous studies [17,28]. Cav-1, a structural protein in caveolae, is required
for the formation of calcifying EVs [7]. Additionally, it
has been shown that disassembly of caveolae buffers sudden increases in stress on the plasma membrane [15].
However, the effect of mechanical stimulation on calcifying EV formation in VSMCs has not been reported.
To address this knowledge gap, we investigated in vitro
behavior of VSMCs in response to mechanical stimulation and showed that mechanical stretch regulates Cav-1

trafficking and its redistribution from VSMCs into EVs.
Further, EVs from VSMCs exposed to mechanical stretch
have increased mineralization potential.
Lipid rafts are distinct microdomains within the plasma
membrane that are enriched in sphingolipids and cholesterol [29, 30]. This unique lipid composition renders lipid
rafts and caveolae less fluid and more tightly packed than
the surrounding phospholipid bilayer. While both planar
lipid rafts and caveolae domains are involved in signaltransduction, planar lipid rafts form small, transient platforms that float freely within the membrane. In contrast,
caveolae are invaginated flask-shaped structures that arestabilized by the structural protein caveolin. The highShaver et al. BMC Molecular and Cell Biology(2024) 25:8

cholesterol content of caveolae allows them to function
as organized signaling centers that compartmentalize andregulate signaling molecules. The cyclic strain-induced
translocation of Cav-1 from caveolae to non-caveolar
domains observed in our study may have significant
functional consequences by modulating Cav-1-mediatedcell signaling. Upon caveolae disassembly and Cav-1 displacement into other membrane regions, dissociation of
Cav-1 from signaling effectors can then lead to changes
in their activation state. For example, previous studies
have shown that mechanical stimuli trigger caveolae disassembly and activation of Src family kinases and ERK
pathway in a mechanism dependent on Cav-1 redistribution [25, 31]. Our data also suggest that the applicationof mechanical stretch increases total intracellular cholesterol after 24 h, perhaps reflecting a feedback attempt
to recover plasma membrane homeostasis following thesudden addition of cyclic tension. Further investigation
into the specific signaling pathways affected by Cav-1 displacement from caveolae will provide insights into mechanisms of vascular mechanotransduction.
Our results demonstrate that cyclic mechanical stretch
alters Cav-1 distribution in VSMCs compared to nonstretched conditions, reducing intracellular Cav-1 while
increasing secretion into EVs. Additionally, stretch
increased intracellular cholesterol in VSMCs. Thischolesterol accumulation may represent a compensatory response to restore caveolae domains disrupted
by plasma membrane deformation. Further investigation into how forces stimulate caveolin-1 trafficking andcholesterol biosynthesis, as well as mobilization into
EVs using diverse in vitro and ex vivo stretch protocols,
will provide insights into the coordination of these processes. Understanding the links between strain-sensitive
caveolin-1 redistribution, cholesterol metabolism, andsecretion may reveal fundamental mechanisms mediating intercellular communication networks in response tovascular biomechanics.
Previous studies suggested that caveolae endocytosis
occurs in response to plasma membrane damage [32,33]. Our observation of Cav-1 re-localization from theplasma membrane to the cytosol in VSMCs exposedto cyclic mechanical stretch is consistent with caveolae endocytosis induced by membrane strain. VSMCs
exposed to cyclic mechanical stretch do not exhibit
reduced viability compared to non-stretched samples(Supplemental Fig. 1), suggesting that the observed
increase in extracellular Cav-1 is due to active trafficking
within live cells. As noted above, density gradient fractionation of stretched VSMCs revealed a shift of Cav-1
out of buoyant, caveolae-enriched fractions into denser,
non-caveolar lipid domains. This implies that caveolae
may internalize into the cytosolic space at these domains

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upon mechanical perturbation. Dynamin has been identified as a key mediator of caveolae budding and endocytosis [34, 35]. Accordingly, dynamin inhibition reduced
Cav-1 release in EVs from stretched VSMCs, implicating caveolae internalization as a prerequisite for loading
Cav-1 into EVs with cyclic strain. The exosomal marker
CD63 was unchanged in EVs from stretched versus nonstretched VSMCs, indicating distinct regulation of Cav1derived vesicles. Cav-1 has been previously associatedwith regulation of microRNA sorting into microvesicles[36], which bud directly from the plasma membrane ofcells. Future studies in intracellular trafficking and exosome-like secretion of Cav-1-positive EVs may yield new
insight into the regulation of this unique EV population.
The actin cytoskeleton often mediates cellularresponses to mechanical stimulation [37]. Besides theregulation of membrane tension, actin stress fibers
directly associate with caveolae and play a critical rolein the intracellular trafficking, endocytosis and exostosisof vesicles [14, 38]. Rho-A kinase (ROCK) is a memberof the GTPase family and participates in actin filament
polymerization and contraction [39]. Additionally, ROCK
contains a Cav-1-binding domain [40]. ROCK inhibition
reduced actin polymerization in VSMCs, yet increased
Cav-1 release in extracellular vesicles. These results
reveal that actin dynamics influence Cav-1 externalization, though the mechanisms remain unclear. Ongoingstudies are needed to delineate how alterations in actin
polymerization and contractility modulate EV Cav-1
loading, including the potential contributions of actin
enhancement pathways. Elucidating the cytoskeletal
control of Cav-1 trafficking will provide broader insights
into how the actin network integrates mechanical cues todirect intercellular communication through extracellular
vesicles.
Vascular calcification often associates with hyperphosphatemia [41]. Our results revealed that EVs released
from stretched VSMCs have higher potential to form
calcium-phosphate mineral when they are placed in ahigh phosphate environment. Annexin V is a phospholipid-binding protein that has previously been implicatedin mineral nucleation within EVs [27]. We detected anincrease in annexin V protein in EVs from VSMCs subjected to 10% strain. The 3D collagen hydrogel platform
allowed us to visualize mineral nucleation as an in vitro
model of vascular calcification. Quantitative image
analysis revealed increased numbers of mineral nucleation sites and total area covered by mineral in hydrogels
treated with EVs from cyclically stretched VSMCs. Thesebiomimetic calcification assays confirm that mechanicalforces enhance the release of EVs with greater propensity
to induce calcium phosphate deposition, complementing our cell culture data. Together, these data suggest

Shaver et al. BMC Molecular and Cell Biology(2024) 25:8

that mechanical stimulation leads to release of EVs withelevated mineralization potential. Future studies will
directly assess the causal role of altered Cav-1 trafficking
in promoting the increased mineralization potential.

Conclusion
In conclusion, this study shows that cyclic stretch
alters Cav-1 distribution and enhances release in EVs
from VSMCs. Stretch-induced caveolin-1 mobilization involves caveolae endocytosis. Moreover, EVs fromstretched VSMCs exhibit increased mineralization
potential. A proposed mechanism diagram revealing theregulation of cyclic mechanical stretch on Cav-1 redistribution and extracellular vesicle release in vascularsmooth muscle cells is shown in Fig. 7. Further researchon links between biomechanics, Cav-1 dynamics, andEV function will provide insights into vascular diseaseprogression.
Materials and methods
Cell culture and mechanical stretch

Primary mouse and human VSMCs (ATCC, Manassas, VA) were cultured in Dulbecco’s Modified Eagle’s
Medium (DMEM) supplemented with 10% fetal bovine
serum and 1% penicillin–streptomycin at 37 °C in a

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humidified atmosphere of 5% CO2 and 95% air. Afterreaching 80–90% confluence, VSMCs between passages 3–8 were used for experiments. In addition tomouse vascular smooth muscle cells, a human cell line
(ATCC, Manassas, VA) was utilized for RNA interference and osteogenic differentiation experiments in order
to translate and validate key findings across species. To
apply mechanical stretch, VSMCs with the cell density of
2 × ­105 cells/well were seeded onto collagen type I precoated 6-well Bioflex® plates (Flexcell International Crop,
Burlington, NC) in the presence of DMEM, 1% penicillin/
streptomycin and 10% EVs deprived serum. EV-deprived
serum was obtained by ultracentrifugation at 100,000 × g
for 15 h, after which the supernatant was collected foruse. To apply 10% equibiaxial mechanical stretch (0.5 Hz,72 h) on cultured VSMCs, a Flexcell® FX-5000 T tension
system (Flexcell International Corp.) was utilized. Nonstretched VSMCs seeded on 0.2% gelatin-coated tissue
culture plastic plates were used to establish a common
baseline for comparison between the groups exposed
to different levels of stretch. To determine the viability of VSMCs following prescribed culture regimen, weused a commercially available assay kit (Abcam, Cambridge, United Kingdom). The live cell dye penetrates
intact, live cells and produces a fluorescence signal (Ex/Fig. 7 Proposed mechanism of cyclic mechanical stretch-induced caveolin-1 trafficking in vascular smooth muscle cells. Mechanical stimulation
promotes caveolae endocytosis in a dynamin-dependent process, reducing caveolin-1 levels in the plasma membrane. Caveolin-1 is subsequently
trafficked to the cytosol and incorporated into extracellular vesicles, leading to increased release. The extracellular vesicles from stretched cells
exhibit increased alkaline phosphatase (ALP) activity. RhoA kinase (ROCK) mediates actin cytoskeleton remodeling in response to stretch, which
regulates caveolin-1 mobilization

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Em = 485/530 nm) following hydrolysis by active intracellular esterases. The fluorescent dead cell dye (Ex/Em = 495/635 nm) is impermeable to live cells but entersdamaged cells and binds to nucleic acids. Following theaddition of the dyes according to the manufacturer’s
instructions, cells were imaged with a Zeiss Axioscope
upright fluorescence microscope. Live and dead cells
were counted using ImageJ and used to calculate overall
viability.
The following specific inhibitors were used for the prescribed treatments: Dynasore (40 µM; APExBIO, Houston, TX) as GTPase inhibitor of dynamin, and Y-27632
dihydrochloride (10 µM; Biotechne, Bristol, United Kingdom) for RhoA kinase inhibition.

raft isolation kit (Sigma-Aldrich) according to the manufacturer’s instructions. Briefly, cultured VSMCs werelysed in ice-cold lysis buffer containing 1% Triton X-100
and homogenized. The lysates were combined with anequal volume of 60% OptiPrep density gradient medium
and overlaid with layers of 35% and 5% OptiPrep ingradient buffer. The samples were ultracentrifuged at
200,000 × g for 4 h at 4 °C to allow separation of detergent-soluble and insoluble fractions across the gradient.
Detergent-resistant membranes enriched in caveolae/
raft components floated to the 5–35% interface (1–7 fractions) and were collected. These buoyant membrane fractions were confirmed to be enriched in canonical lipid
raft markers by immunoblotting of Cav-1.

Osteogenic stimulation

Cellular cholesterol measurment

In order to induce a pro-calcific phenotype, VSMCs were
cultured in osteogenic media for 7 days prior to applying
10% mechanical stretch, in order to induce a pro-calcificphenotype. The osteogenic media consisted of complete control media supplemented with dexamethasone(10 nM; Sigma), L-ascorbic acid (100 μM; Sigma), andβ-glycerolphosphate (10 mM; Sigma).
Western blot analysis

VSMCs lysates were prepared in ice-cold RIPA Buffer.
EVs were isolated from the conditioned media of VSMCs
via centrifugation at 1000 g for 5 min to remove celldebris followed by ultracentrifugation at 100,000 g for
60 min at 4 °C using an Optima Max-TL ultracentrifuge(Beckman Coulter, Brea, CA). Isolated EVs were lysed inprotein lysis buffer. Following the BCA protein concentration assessment, equal amounts of the lysates wereseparated with 10% SDS–polyacrylamide gel electrophoresis and then transferred onto nitrocellulose membranes(Bio-Rad, Hercules, CA). Due to lack of appropriate loading control for the EVs, Ponceau S was used to confirm
equal protein loading. Membranes were blocked for 1 h
at room temperature in casein blocking buffer in TBST
(Sigma-Aldrich, St. Louis, MO) and then 1–2 h at room
temperature with primary antibodies for Caveolin-1(1:500; Abcam, Cambridge, United Kingdom), Annexin
V (1:500; Abcam, Cambridge, United Kingdom) or CD63
(1:500; Abcam, Cambridge, United Kingdom). Following washing, the species appropriate secondary antibody(1:10,000; LI-COR, Lincoln, NE) was added for 1 h atroom temperature. Visualization was performed using an
Odyssey®CLx (LI-COR, Lincoln, NE) and band densities
were analyzed using Image Studio® (LI-COR).
Caveolae/lipid raft isolation

Isolation of detergent-resistant membranes enriched incaveolae and lipid rafts was performed using a caveolae/

Cellular lipids and cholesterol were extracted from
cultured VSMCs using an organic solvent-based
method. Briefly, VSMCs were homogenized in a 3:2
hexane:isopropanol solution to lyse cell membranes andextract lipids. The homogenates were centrifuged to separate the aqueous and organic phases. The upper organic
phase containing lipids was transferred to a clean microcentrifuge tube and dried under vacuum to recover total
cellular lipids. Immediately prior to quantification, thedried lipids were resuspended in isopropanol. Total cholesterol content was measured employing Cholesterol E
kit (Fujifilm Wako Diagnostics) enzymatic colorimetricassay kit according to the manufacturer’s protocol.
Immunofluorescence analysis

VSMCs were fixed with 4% formalin for 10–15 min andpermeabilized by 0.1% Triton X-100 (Fisher scientific,
Waltham, MA) for 10 min. Non-specific binding sites
were blocked with 0.1 mM glycine in 1% BSA for 30 min
at room temperature. Next, cells were incubated withcaveolin-1 primary antibody (1:200) for 2 h at room
temperature, washed with 1X PBS, and incubated withsecondary antibody Alexa Fluor® 594 (1:400; Abcam,
Cambridge, United Kingdom) for 1 h at room temperature. Cells were co-stained with Phalloidin-iFluor™ 488
Conjugate (1:1000; Cayman chemical, Ann Arbor, MI) for
30 min. The stained cells were mounted in Permount™
Mounting Medium (Fisher scientific) and imaged usinga laser scanning confocal microscope (Eclipse Ti, Nikon,
Minato City, Tokyo, Japan).
RNA interference

To silence caveolin-1 expression, 50 nm siRNA against
caveolin-1 (s2448, Life Technology) and non-targetingsiRNA (ON-TARGET Non-Targeting Pool, Thermo
Scientific) was transfected into VSMCs using (Thermo

Shaver et al. BMC Molecular and Cell Biology(2024) 25:8

Scientific). Transfection was performed 48 h prior tomechanical stretch exposure.
Mineral formation assay

In order to measure the real-time mineral formationof EVs, we used an assay originally developed to study
mineral formation from growth plate cartilage EVs [42].
For kinetic assay, 1% (v/v) of 300 mM N
­ aH2PO4 (SigmaAldrich, St. Louis, MO) was added to 200 µl of EVs stock
solution of each sample and suspensions were placed
into 96 well plates and incubated at 37 °C in a Synergy
HT microplate reader (Biotek, Winooski, VT) for 16 h.
The absorbency reading was recorded at 340 nm at 1-min
interval.
Collagen hydrogel experiments

Collagen hydrogels were prepared by mixing high-concentration rat tail collagen with DMEM in a 1:19 ratio
and neutralizing the mixture to pH 7–8. The neutralized collagen solution was then added to chamberedcoverglass wells (LAB-TEK). The conditioned mediaof VSMCs, containing EVs, were added to the collagennetwork and incubated at 37 °C for the indicated time
period. One day before imaging, a near-infrared-based
bisphosphonate calcium tracer (OsteoSense 680, Perkin
Elmer) was added to the hydrogels and incubated overnight at 37 °C. The resulting aggregation and calcification
processes were imaged by fluorescence microscopy (Ziess
AXIO). Quantitative image analysis of the captured fluorescence micrographs was performed using ImageJ software (NIH).
Alkaline phosphatase activity assay

Alkaline phosphatase (ALP) activity in cells and EVs wasmeasured using a colorimetric assay kit (Abcam) according to the manufacturer’s protocol. Briefly, cell lysates
were collected in the supplied buffer and centrifuged toremove insoluble material. The supernatants were added
to a 96-well plate at a volume of 80 μL per well after dilution with the provided assay buffer. Diluted p-nitrophenyl phosphate (pNPP) substrate was then added to eachsample well and the plate was incubated at 25 °C protected from light for 1 h to allow the enzymatic reaction
to occur. The reaction was stopped by addition of a stop
solution. Finally, the absorbance was measured at 405 nm
using a microplate reader to quantify ALP activity.
Statistical analysis

Each experiment was performed for at least three biological replications, and all data were presented asmean ± SD. Student’s t-test was used to analyze twogroup comparisons. Multiple group comparisons wereconducted by one-way ANOVA. All statistical tests were

Page 11 of 12

calculated using GraphPad Prism 9.00 (GraphPad Software). A P-value less than 0.05 was considered statistically significant.
Abbreviations
ALP	Alkaline phosphatase
AnxV	Annexin V
Cav-1	Caveolin 1
EV	Extracellular vesicle
ROCK	RhoA Kinase
VSMCs	Vascular smooth muscle cells

Supplementary Information
The online version contains supplementary material available at https://​doi.​
org/​10.​1186/​s12860-​024-​00504-w.
Supplementary Material 1.
Authors’ contributions
M.S. helped design the study, carried out all experimental procedures, performed data analyses, and drafted the original version of the manuscript. K.G.
helped with the collagen hydrogel assay and edited the revised manuscript.
K.K. performed cell viability analyses and edited the revised manuscript. J.D.H.
conceived of the original idea for the study, helped design the study, aided indata analyses, and edited the manuscript.
Funding
This work was supported by the Florida Heart Research Foundation (StopHeart Disease Researcher of the Year Award to J.D.H and a Doctoral Student
Grant to M.S.) and the National Heart, Lung, and Blood Institute of the National
Institutes of Health (1R01HL160740 to J.D.H.). The content is solely the responsibility of the authors and does not necessarily represent the official views of
the National Institutes of Health.
Availability of data and materials
The complete raw datasets employed and/or examined in this study will bemade available upon reasonable request to the corresponding author.

Declarations
Ethics approval and consent to participate
Not applicable.
Consent for publication
Not applicable.
Competing interests
The authors declare no competing interests.
Received: 30 August 2023 Accepted: 1 March 2024

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