Vascular Smooth Muscle Cell Stiffness Research Articles

Cystathionine γ lyase Attenuates Vascular Smooth Muscle Cell Senescence via Foxm1-Gas1 Pathway to Mediate Arterial Stiffness.

Aims Arterial stiffness, a hallmark of vascular aging, significantly contributes to hypertension and impaired organ perfusion. Vascular smooth muscle cell (VSMC) dysfunction, particularly VSMC senescence and its interaction with stiffness, is crucial in the pathogenesis of arterial stiffness. Although hydrogen sulfide (H2S) and its key enzyme cystathionine γ-lyase (CSE) are known to play roles in cardiovascular diseases, their effects on arterial stiffness are not well understood. Methods & Results First, we observed a downregulation of CSE/H2S in the aortic media during biological aging and Angiotensin II (AngII)-induced aging. The VSMC-specific CSE knockout mice were created by loxp-cre (Tagln-cre) system, and which exacerbated AngII-induced aortic aging and stiffness in vivo and VSMC senescence and stiffness in vitro. Conversely, the CSE agonist norswertianolin mitigated these effects. Next, we identified growth arrest-specific 1 (Gas1) as a crucial target of CSE/H2S and found it to be a downstream target gene of forkhead box protein M1 (Foxm1). siRNA knockdown Foxm1 increased Gas1 transcription and reduced the protective effects of H2S on VSMC senescence and stiffness. Finally, we demonstrated that CSE/H2S sulfhydrates Foxm1 at the C210 site, regulating its nuclear translocation and activity, thus reducing VSMC senescence and stiffness. Innovation Our findings highlight the protective role of CSE/H2S in arterial stiffness, emphasizing the novel contributions of CSE, Gas1, and Foxm1 to VSMC senescence and stiffness. Conclusion Endogenous CSE/H2S in VSMCs reduces VSMC senescence and stiffness, thereby attenuating arterial stiffness and aging, partly through sulfhydration-mediated activation of Foxm1 and subsequent inhibition of Gas1 signaling pathways.

ITIH4 reversed the effects of thrombin on VSMCs stiffness via JNK and ERK signaling pathway

Vascular smooth muscle cell (VSMCs) is one of the important cell types in artery. VSMCs stiffening may regulate vascular stiffness and contribute to the development of vulnerable plaques. Thrombin, an enzyme in coagulation system, is involved in pathological processes of atherosclerosis. Inter-alpha-trypsin inhibitor heavy chain 4 (ITIH4) plays an important role in regulating inflammation and may have cardiovascular protective effect. Therefore, the elucidation of the mechanisms underlying ITIH4-mediated VSMCs stiffening helps to provide new ideas and potential targets for the diagnosis and treatment of atherosclerosis. In this study, we used specific ITIH4 expression vector and siRNA methods to transfect VSMCs. Our results found that ITIH4 expression increased VSMCs stiffness, meanwhile, ITIH4 siRNA decreased VSMCs stiffness. ITIH4 increased acetylated α-tubulin and inhibited ERK1/2 and JNK, but not P38 MAPK. ERK inhibitor (PD98059) or JNK inhibitor (SP600125) treatment increased acetylated α-tubulin expression and cell stiffness in VSMCs. ITIH4 was downregulated by thrombin treatment, ITIH4 partly reversed the effect of thrombin on acetylated α-tubulin and VSMCs stiffness. These results indicated that ITIH4 regulated acetylated α-tubulin expression in VSMCs and was against the effects of thrombin on VSMCs stiffness. JNK and ERK signaling pathways were proved to participate in this process.

Vascular Smooth Muscle Cell Mechanical Stretch Modulates Tissue Transglutaminase Activity and Cytoskeletal Dynamics

Vascular smooth muscle cells (VSMCs) are exposed to a wide range of mechanical stimuli in both physiological and pathological conditions. These mechanical stimuli are converted into biochemical signals that regulate VSMC contractility and cytoskeletal structures in a process known as mechanotransduction. Thus, the ability of VSMCs to sense and respond to mechanical signals is essential for proper regulation of their structural characteristics and the modulation of vascular tone. We had previously shown that increased tissue transglutaminase 2 (TG2) activity modulates actin dynamics via alterations of the phosphorylation state of cofilin leading to VSMC stiffening and remodeling of the vascular wall. However, whether mechanical forces act through TG2 to modulate VSMC stiffness remains unknown. Herein, we tested the hypothesis that mechanical stretch increases VSMC stiffness by increasing TG2 activity, reducing cofilin activity, and increasing actin cytoskeletal stress fiber formation. To this end, immortalized human aortic vascular smooth muscle cells (HAoSMCs) were cultured and seeded on Uniflex culture plates. HAoSMCs were left unstretched or exposed to cyclic uniaxial stretch using a Flexcell tension system with 11% elongation at 1 Hz for 6 hrs. HAoSMCs were treated without or with the nitric oxide (NO) donor s-nitrosoglutathione (400μmol/L) or cystamine (200μmol/L) under stretched and unstretched conditions, as NO and cystamine are known inhibitors of TG2. All differences reported herein are significant at p<0.05. Our results show that cyclic uniaxial stretch for 6 hrs increases VSMC TG2 content and activity, actin stress fibers (polymerization), and phospho-cofilin/cofilin ratios (cofilin inactivation), relative to the unstretched condition. Notably, the increase in TG2 activity and F-actin content occurring in response to stretch was inhibited by the NO donor or cystamine. As it has been previously shown that cellular stiffness is associated with stress fiber formation, this work suggests that stretch-induced cellular stiffening is potentially mediated by TG2 activity in VSMC. National Institutes of Health grant: R01HL153264 to LM-L and JP. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Abstract 454: The Effect Of High-fat Diet On Vascular Smooth Muscle Cell Mechanics And Migration In Apoe -/- Mice

Atherosclerosis remains a leading cause of cardiovascular diseases, which cause causes about 30% of global death. Vascular smooth muscle cells (VSMCs) are the major cellular components of arterial wall. VMSCs play critical role in the atherosclerosis development by phenotypic switching from a contractile phenotype to a synthetic phenotype, migration from media to intima of arterial wall, and deposition as foam cells in atherosclerotic plaque. In this study, we tested the effect of high-fat diet on the VSMC mechanics by comparing the mechanics and migration of VSMCs isolated from apolipoprotein-E knockout (ApoE -/- ) and wild type (WT) mice. Atomic force microscope (AFM) was employed to test cell adhesion and stiffness of VSMCs. AFM was also used to examine live VSMC submembranous cytoskeleton organization. A data-driven mathematical models-based image and signal processing software was used to analyze AFM data. Two-way ANOVA was used to test statistical significance for all experiments. The results demonstrated significant differences in the actin cytoskeletal organization and cell mechanics between ApoE -/- VSMCs and WT cells upon high-fat diet. However, no significant difference in cell stiffness was observed between normal diet-fed ApoE -/- and WT VSMCs. These interesting findings on the effect of high-fat diet on VSMC mechanics will improve our knowledge of the mechanism underlying the contribution of VSMC in the development of atherosclerosis.

Exploring the difference in the mechanics of vascular smooth muscle cells from wild-type and apolipoprotein-E knockout mice.

Atherosclerosis-related cardiovascular diseases are a leading cause of mortality worldwide. Vascular smooth muscle cells (VSMCs) comprise the medial layer of the arterial wall and undergo phenotypic switching during atherosclerosis to a synthetic phenotype capable of proliferation and migration. The surrounding environment undergoes alterations in extracellular matrix (ECM) stiffness and composition and an increase in cholesterol content. Using an atherosclerotic murine model, we analyzed how the mechanics of VSMCs isolated from Western diet-fed apolipoprotein-E knockout (ApoE-/-) and wild-type (WT) mice were altered during atherosclerosis. Increased stiffness of ApoE-/- VSMCs correlated with a greater degree of stress fiber alignment, as evidenced by atomic force microscopy (AFM)-generated force maps and stress fiber topography images. On type-1 collagen (COL1)-coated polyacrylamide (PA) gels (referred to as substrate) of varying stiffness, ApoE-/- VSMCs had lower adhesion forces to COL1 and N-cadherin (N-Cad) compared with WT cells. ApoE-/- VSMC stiffness was significantly greater than that of WT cells. Cell stiffness increased with increasing substrate stiffness for both ApoE-/- and WT VSMCs. In addition, ApoE-/- VSMCs showed an enhanced migration capability on COL1-coated substrates and a general decreasing trend in migration capacity with increasing substrate stiffness, correlating with lowered adhesion forces as compared with WT VSMCs. Altogether, these results demonstrate the potential contribution of the alteration in VSMC mechanics in the development of atherosclerosis.

Age and Blood Pressure Contribute to Aortic Cell and Tissue Stiffness Through Distinct Mechanisms.

Aortic stiffening is strongly associated with both aging and hypertension, but the underlying mechanisms remain unclear. We hypothesized that aging-induced aortic stiffness is mediated by a mechanism differing from hypertension. We conducted comprehensive in vivo and in vitro experiments using multiple rat models to dissect the different mechanisms of aortic stiffening mediated by aging and hypertension. A time-course study in spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) normotensive rats showed more pronounced aging-associated aortic stiffening in SHR versus WKY. Angiotensin II-induced hypertension was associated with more significant aortic stiffening in older versus young WKY rats. Hypertension aggravated aging effects on aortic wall thickness and extracellular matrix content, indicating combinational effects of aging and hypertension on aortic stiffening. Intrinsic stiffness of isolated aortic vascular smooth muscle cells (VSMCs) increased with age in WKY rats, although no significant difference between older SHR and older WKY VSMCs was observed in 2-dimensional culture, reconstituted 3-dimensional tissues were stiffer for older SHR versus older WKY. A selective inhibitor that reduced hypertension-mediated aortic stiffening did not decrease age-related stiffening in aortic VSMCs and aortic wall. Integrin β1 and SM22 (smooth muscle-specific SM22 protein) expression were negligibly changed in WKY VSMCs during aging but were markedly increased by hypertension in older versus young WKY VSMCs. A notable shift of filamin isoforms from B to A was detected in older WKY VSMCs. Our results indicate distinct mechanisms mediating aging-associated aortic VSMC and vessel stiffness, providing new insights into aortic stiffening and the pathogenesis of hypertension in the elderly.

Substrate Stiffness Modulates Normal and Diabetic Coronary Smooth Muscle Cell Stiffness

We have previously reported that coronary resistance microvessels (CRMs) from 16‐week‐old type 2 diabetic (db/db) mice were less stiff compared to normal heterozygous Db/db controls, a finding that was driven by less stiff coronary vascular smooth muscle cells (VSMCs). Because the surrounding extracellular matrix (ECM) substrates in vivo may impact cellular properties such as elastic modulus (a measure of stiffness), the aim of this study was to test the hypothesis that modulating substrate stiffness correspondingly modulates the elastic modulus of normal and diabetic coronary VSMCs in vitro. In this study, we plated low‐passage coronary VSMCs isolated from Db/db and db/db mice onto 60 mm polyacrylamide dishes (Matrigen) that were of varying stiffnesses – 4 kPa, 25 kPa, and 50 kPa (n=4 per group). Cells were serum starved for 24 hours, and then cell stiffness was measured by atomic force microscopy (AFM). We observed a stepwise increase in cellular elastic modulus with each increase in polyacrylamide substrate stiffness in both the normal and diabetic coronary VSMCs (Db/db 4 kPa = 3.39±0.48 kPa, Db/db 25 kPa = 5.46±0.78 kPa, Db/db 50 kPa = 7.13±0.29 kPa; db/db 4 kPa = 2.49±0.23, db/db 25 kPa = 5.00±0.52 kPa, db/db 50 kPa = 5.58±0.16 kPa, p<0.0001). Similar to what we have previously reported on cells grown on plastic, diabetic coronary VSMCs were consistently less stiff compared to normal at each substrate stiffness. We did not observe statistically significant differences in cellular filamentous (F) actin, globular (G) actin, or the F/G actin ratio on the varying polyacrylamide substrate stiffness for neither Db/db nor db/db coronary VSMCs. However, there was a preliminary trend for lower F actin (Db/db: 1141.00 ± 472.20 AU vs. db/db: 422.50 ± 240.30 AU at 4 kPa, p=0.24, n=2 per group) and F/G actin ratio (Db/db: 4.01 ± 0.92 AU vs. db/db: 2.55 ± 0.35 AU at 4 kPa, p=0.21, n=2 per group) in the db/db VSMCs, corroborating our past data demonstrating that db/db coronary VSMCs are less stiff compared to normal Db/db coronary VSMCs. Overall, these data show that substrate stiffness correspondingly modulates normal and diabetic coronary VSMC stiffness.

Is It Good to Have a Stiff Aorta with Aging? Causes and Consequences.

Aortic stiffness increases with advancing age, more than doubling during the human life span, and is a robust predictor of cardiovascular disease (CVD) clinical events independent of traditional risk factors. The aorta increases in diameter and length to accommodate growing body size and cardiac output in youth, but in middle and older age the aorta continues to remodel to a larger diameter, thinning the pool of permanent elastin fibers, increasing intramural wall stress and resulting in the transfer of load bearing onto stiffer collagen fibers. Whereas aortic stiffening in early middle age may be a compensatory mechanism to normalize intramural wall stress and therefore theoretically "good" early in the life span, the negative clinical consequences of accelerated aortic stiffening beyond middle age far outweigh any earlier physiological benefit. Indeed, aortic stiffness and the loss of the "windkessel effect" with advancing age result in elevated pulsatile pressure and flow in downstream microvasculature that is associated with subclinical damage to high-flow, low-resistance organs such as brain, kidney, retina, and heart. The mechanisms of aortic stiffness include alterations in extracellular matrix proteins (collagen deposition, elastin fragmentation), increased arterial tone (oxidative stress and inflammation-related reduced vasodilators and augmented vasoconstrictors; enhanced sympathetic activity), arterial calcification, vascular smooth muscle cell stiffness, and extracellular matrix glycosaminoglycans. Given the rapidly aging population of the United States, aortic stiffening will likely contribute to substantial CVD burden over the next 2-3 decades unless new therapeutic targets and interventions are identified to prevent the potential avalanche of clinical sequelae related to age-related aortic stiffness.

Vascular smooth muscle cell-specific miRNA-214 knockout inhibits angiotensin II-induced hypertension through upregulation of Smad7.

Vascular remodeling is a prominent trait during the development of hypertension, attributable to the phenotypic transition of vascular smooth muscle cells (VSMCs). Increasing studies demonstrate that microRNA plays an important role in this process. Here, we surprisingly found that smooth muscle cell-specific miR-214 knockout (miR-214 cKO) significantly alleviates angiotensin II (Ang II)-induced hypertension, which has the same effect as that of miR-214 global knockout mice in response to Ang II stimulation. Under the treatment of Ang II, miR-214 cKO mice exhibit substantially reduced systolic blood pressure. The vascular medial thickness and area in miR-214 cKO blood vessels were obviously reduced, the expression of collagen I and proinflammatory factors were also inhibited. VSMC-specific deletion of miR-214 blunts the response of blood vessels to the stimulation of endothelium-dependent and -independent vasorelaxation and phenylephrine and 5-HT induced vasocontraction. In vitro, Ang II-induced VSMC proliferation, migration, contraction, hypertrophy, and stiffness were all repressed with miR-214 KO in VSMC. To further explore the mechanism of miR-214 in the regulation of the VSMC function, it is very interesting to find that the TGF-β signaling pathway is mostly enriched in miR-214 KO VSMC. Smad7, the potent negative regulator of the TGF-β/Smad pathway, is identified to be the target of miR-214 in VSMC. By which, miR-214 KO sharply enhances Smad7 levels and decreases the phosphorylation of Smad3, and accordingly alleviates the downstream gene expression. Further, Ang II-induced hypertension and vascular dysfunction were reversed by antagomir-214. These results indicate that miR-214 in VSMC established a crosstalk between Ang II-induced AT1R signaling and TGF-β induced TβRI /Smad signaling, by which it exerts a pivotal role in vascular remodeling and hypertension and imply that miR-214 has the potential as a therapeutic target for the treatment of hypertension.

Degradation of Premature-miR-181b by the Translin/Trax RNase Increases Vascular Smooth Muscle Cell Stiffness.

[Figure: see text].

ANGIOTENSIN II-INDUCED ER STRESS RESPONSE CONTRIBUTES TO PROFIBROTIC VSMC PHENOTYPE AND SUBSEQUENT HYPERTENSIVE ARTERIAL STIFFNESS

Objective: The renin-angiotensin-aldosterone system is known to play a pivotal role in human hypertension, and specifically angiotensin II (Ang II) is believed to mediate arterial stiffness through maladaptive responses from vascular smooth muscle cells (VSMCs). The objective of this study was to elucidate Ang II activation of the endoplasmic reticulum unfolded protein response (ER UPR) in VSMCs, how this contributes to hypertrophic and fibrotic responses, and how inhibition of ER UPR may mitigate increased aortic pulse propagation velocity (PPV, a surrogate measurement for aortic stiffness). Design and method: ApoE-/- mice were infused with 1 ug/kg/min angiotensin II for 14 days with or without 1 mg/mL 4-PBA For in vitro studies, rat VSMCs were explanted and used from P4-P8. 4-PBA 30 min pretreatment at 1 mM before Ang II 48 hr stimulation or adeno GRP78 infection used to assess chaperoning effect on sirius red collagen assay, THP-1 adhesion, and protein aggregate accumulation. Results: Chronic 14 day infusion of Ang II in apoE-/- mice leads to increased PPV from 0.06 m/s to 0.12 m/s, whereas supplementing drinking water with 1 mg/mL chemical ER chaperone 4-PBA mitigates this induction (PPV = 0.055 m/s), P = 0.039. Fibrosis of the heart, kidney and aorta were reduced with 4-PBA treatment as well. ER stress markers pIRE1a and XBP1S were activated in the thoracic aorta by Ang II infusion and inhibited by 4-PBA. Preamyloid oligomer accumulation induced by Ang II in the aorta was also reduced by 4-PBA. Rat VSMCs stimulated with Ang II were stained with aggregate dye Proteostat, which was increased with 48 hour Ang II treatment, and inhibited with 4-PBA pretreatment (P < 0.05). To evaluate VSMC stiffness promoting phenotype, collagen production and immune cell adhesion were assessed by Sirius Red assay and THP-1 adhesion, respectively. Both inductions by Ang II were decreased by 4-PBA. Overexpression of GRP78 significantly mitigated aggregate formation (P < 0.05) and immune cell infiltration (P < 0.05). Conclusions: Ang II induced pulse pressure and vascular amyloid deposition is decreased by chaperoning nascent protein folding. Targeting GRP78 to reduce ER UPR activation and subsequent disturbances in proteostasis may be a therapeutic strategy for aortic stiffness.

Stiffness of Vascular Smooth Muscle Cells from Monkeys Studied using Atomic Force Microscopy

Vascular smooth muscle cells (VSMC) are the main cellular components of blood vessel walls and bear external mechanical forces caused by blood flow and pressure. In this report, we have verified the following hypothesis through experiments: The increase in VSMC stiffness may be mainly due to changes in vascular stiffness due to aging. Although aging enhances the stiffness and adhesion of VSMC, there is no significant difference in apparent elastic modulus and adhesion between the VSMC obtained by male and female. The effect of aging through the ECM-integrin-cytoskeleton axis is related to increased VSMC stiffness and matrix adhesion rather than gender.

Abstract MP22: GRP78 Protects Against Angiotensin II-induced Protein Aggregation, Profibrotic VSMC Phenotype, And Vascular Remodeling

Arterial stiffening is a critical risk factor contributing to the development of cardiovascular disease and end organ damage. Vascular smooth muscle cell (VSMC) hypercontractility, vessel fibrosis, and proinflammatory responses are involved. The renin angiotensin system plays critical roles in the development of VSMC stiffness phenotype, as well as CVD pathophysiology. Angiotensin II (Ang II) stimulation induces these stiffness-promoting mechanisms, however, the involvement of dysfunctional proteostasis governing Ang II contribution to vascular stiffness remains underdefined. While endoplasmic reticulum (ER) stress has been implicated in the development of arterial stiffness and hypertension, it is unknown whether the protein aggregation potentially due to maladapted unfolded protein response (UPR) in VSMCs is involved. We hypothesize AngII induced ER stress initiates protein misfolding and effects VSMC stiffness phenotype. We identify a significant increase in protein aggregate size (P<0.001: from 1,000 to 4,500 pixel/aggregate) and number per cell (P<0.01: from 20 to 100 aggregate #/cell) in rat VSMCs stimulated with AngII, which is attenuated with adenoviral overexpression of resident ER chaperone, GRP78 (P<0.01: 2000 pixels/aggregate and P<0.05: 25 aggregate #/cell). Furthermore, UPRosome associated IRE1α phosphorylation induced at 3 and 6 h following AngII stimulation is reduced in GRP78 overexpressing VSMCs (P<0.05), which may contribute to reduced monocyte adhesion (P<0.001), assessed utilizing THP-1 cells. In mice, pharmacological chaperone 3-HNA reduced AngII induced vascular hypertrophy (P=0.056), cardiac perivascular fibrosis (P=0.065), and pulse propagation velocity from 0.35 m/s to 0.23 m/s (P<0.05). A trend in pre-amyloid oligomer accumulation was seen in the medial layer of thoracic aortas following 2 weeks of AngII infusion, which was reduced with 3-HNA treatment. Similar findings were investigated in a SM22α cre-GRP78 mouse model, where a trend in reduction of these parameters are also seen. Overall, GRP78 regulation over VSMC proteostasis influences vascular stiffness phenotype and therefore, these findings may elude new pharmacological intervention for arterial stiffness and aging associated CVD.

Reduced stiffness and augmented traction force in type 2 diabetic coronary microvascular smooth muscle.

Type 2 diabetic (T2DM) coronary resistance microvessels (CRMs) undergo inward hypertrophic remodeling associated with reduced stiffness and reduced coronary blood flow in both mice and pig models. Since reduced stiffness does not appear to be due to functional changes in the extracellular matrix, this study tested the hypothesis that decreased CRM stiffness in T2DM is due to reduced vascular smooth muscle cell (VSMC) stiffness, which impacts the traction force generated by VSMCs. Atomic force microscopy (AFM) and traction force microscopy (TFM) were conducted on primary low-passage CRM VSMCs from normal Db/db and T2DM db/db mice in addition to low-passage normal and T2DM deidentified human coronary VSMCs. Elastic modulus was reduced in T2DM mouse and human coronary VSMCs compared with normal (mouse: Db/db 6.84 ± 0.34 kPa vs. db/db 4.70 ± 0.19 kPa, P < 0.0001; human: normal 3.59 ± 0.38 kPa vs. T2DM 2.61 ± 0.35 kPa, P = 0.05). Both mouse and human T2DM coronary microvascular VSMCs were less adhesive to fibronectin compared with normal. T2DM db/db coronary VSMCs generated enhanced traction force by TFM (control 692 ± 67 Pa vs. db/db 1,507 ± 207 Pa; P < 0.01). Immunoblot analysis showed that T2DM human coronary VSMCs expressed reduced β1-integrin and elevated β3-integrin (control 1.00 ± 0.06 vs. T2DM 0.62 ± 0.14, P < 0.05 and control 1.00 ± 0.49 vs. T2DM 3.39 ± 1.05, P = 0.06, respectively). These data show that T2DM coronary VSMCs are less stiff and less adhesive to fibronectin but are able to generate enhanced force, corroborating previously published computational findings that decreasing cellular stiffness increases the cells' ability to generate higher traction force.NEW & NOTEWORTHY We show here that a potential causative factor for reduced diabetic coronary microvascular stiffness is the direct reduction in coronary vascular smooth muscle cell stiffness. These cells were also able to generate enhanced traction force, validating previously published computational models. Collectively, these data show that smooth muscle cell stiffness can be a contributor to overall tissue stiffness in the coronary microcirculation, and this may be a novel area of interest for therapeutic targets.

Statin-mediated cholesterol depletion exerts coordinated effects on the alterations in rat vascular smooth muscle cell biomechanics and migration.

This study demonstrates and evaluates the changes in rat vascular smooth muscle cell biomechanics following statin-mediated cholesterol depletion. Evidence is presented to show correlated changes in migration and adhesion of vascular smooth muscle cells to extracellular matrix proteins fibronectin and collagen. Concurrently, integrin α5 expression was enhanced but not integrin α2. Atomic force microscopy analysis provides compelling evidence of coordinated reduction in vascular smooth muscle cell stiffness and actin cytoskeletal orientation in response to statin-mediated cholesterol depletion. Proof is provided that statin-mediated cholesterol depletion remodels total vascular smooth muscle cell cytoskeletal orientation that may additionally participate in altering ex vivo aortic vessel function. It is concluded that statin-mediated cholesterol depletion may coordinate vascular smooth muscle cell migration and adhesion to different extracellular matrix proteins and regulate cellular stiffness and cytoskeletal orientation, thus impacting the biomechanics of the cell. Not only does cholesterol induce an inflammatory response and deposits in foam cells at the atherosclerotic plaque, it also regulates cellular mechanics, proliferation and migration in atherosclerosis progression. Statins are HMG-CoA reductase inhibitors that are known to inhibit cellular cholesterol biosynthesis and are clinically prescribed to patients with hypercholesterolemia or related cardiovascular conditions. Nonetheless, the effect of statin-mediated cholesterol management on cellular biomechanics is not fully understood. In this study, we aimed to assess the effect of fluvastatin-mediated cholesterol management on primary rat vascular smooth muscle cell (VSMC) biomechanics. Real-time measurement of cell adhesion, stiffness, and imaging were performed using atomic force microscopy (AFM). Cellular migration on extra cellular matrix (ECM) protein surfaces was studied by time-lapse imaging. The effect of changes in VSMC biomechanics on aortic function was assessed using an ex vivo myograph system. Fluvastatin-mediated cholesterol depletion (-27.8%) lowered VSMC migration distance on a fibronectin (FN)-coated surface (-14.8%) but not on a type 1 collagen (COL1)-coated surface. VSMC adhesion force to FN (+33%) and integrin α5 expression were enhanced but COL1 adhesion and integrin α2 expression were unchanged upon cholesterol depletion. In addition, VSMC stiffness (-46.6%) and ex vivo aortic ring contraction force (-40.1%) were lowered and VSMC actin cytoskeletal orientation was reduced (-24.5%) following statin-mediated cholesterol depletion. Altogether, it is concluded that statin-mediated cholesterol depletion may coordinate VSMC migration and adhesion to different ECM proteins and regulate cellular stiffness and cytoskeletal orientation, thus impacting the biomechanics of the cell and aortic function.

Mechanisms of Arterial Stiffening: From Mechanotransduction to Epigenetics.

Arterial stiffness is a major independent risk factor for cardiovascular complications causing isolated systolic hypertension and increased pulse pressure in the microvasculature of target organs. Stiffening of the arterial wall is determined by common mechanisms including reduced elastin/collagen ratio, production of elastin cross-linking, reactive oxygen species-induced inflammation, calcification, vascular smooth muscle cell stiffness, and endothelial dysfunction. This brief review will discuss current biological mechanisms by which other cardiovascular risk factors (eg, aging, hypertension, diabetes mellitus, and chronic kidney disease) cause arterial stiffness, with a particular focus on recent advances regarding nuclear mechanotransduction, mitochondrial oxidative stress, metabolism and dyslipidemia, genome mutations, and epigenetics. Targeting these different molecular pathways at different time of cardiovascular risk factor exposure may be a novel approach for discovering drugs to reduce arterial stiffening without affecting artery strength and normal remodeling.

Vascular smooth muscle stiffness and its role in aging.

Vascular smooth muscle cells (VSMC) are now considered important contributors to the pathophysiological and biophysical mechanisms underlying arterial stiffening in aging. Here, we review mechanisms whereby VSMC stiffening alters vascular function and contributes to the changes in vascular stiffening observed in aging and cardiovascular disease. Vascular stiffening in arterial aging was historically associated with changes in the extracellular matrix; however, new evidence suggests that endothelial and vascular smooth muscle cell stiffness also contribute to overall blood vessel stiffness. Furthermore, VSMC play an integral role in regulating matrix deposition and vessel wall contractility via interaction between the actomyosin contractile unit and adhesion structures that anchor the cell within the extracellular matrix. Aged-induce phenotypic modulation of VSMC from a contractile to a synthetic phenotype is associated with decreased cellular contractility and increased cell stiffness. Aged VSMC also display reduced mechanosensitivity and adaptation to mechanical signals from their microenvironment due to impaired intracellular signaling. Finally, evidence for decreased contractility in arteries from aged animals demonstrate that changes at the cellular level result in decreased functional properties at the tissue level.

Calcium in Vascular Smooth Muscle Cell Elasticity and Adhesion: Novel Insights Into the Mechanism of Action.

Vascular smooth muscle cells (VSMCs) are the predominant cell type in the arterial wall. These cells play a critical role in maintaining vascular homeostasis including vasoconstriction and vasodilatation through active contraction and relaxation. Dysregulation of VSMC function alters the response of blood vessels to mechanical stress, contributing to the pathogenesis of vascular diseases, particularly atherosclerosis and hypertension. The stiffness of VSMCs is a major regulator of vascular function. Previous studies suggest that intracellular Ca2+ controls the stiffness of VSMCs by a mechanism involving myosin contractile apparatus. More recent studies highlight important functions of cytoskeletal α-smooth muscle actin (α-SMA), α5β1 integrin, and integrin-mediated cell-extracellular matrix (ECM) interactions in Ca2+-dependent regulation of VSMC stiffness and adhesion to the ECM, providing novel insights into the mechanism of calcium action.

Increased mitochondrial NADPH oxidase 4 (NOX4) expression in aging is a causative factor in aortic stiffening

Aging is characterized by increased aortic stiffness, an early, independent predictor and cause of cardiovascular disease. Oxidative stress from excess reactive oxygen species (ROS) production increases with age. Mitochondria and NADPH oxidases (NOXs) are two major sources of ROS in cardiovascular system. We showed previously that increased mitochondrial ROS levels over a lifetime induce aortic stiffening in a mouse oxidative stress model. Also, NADPH oxidase 4 (NOX4) expression and ROS levels increase with age in aortas, aortic vascular smooth muscle cells (VSMCs) and mitochondria, and are correlated with age-associated aortic stiffness in hypercholesterolemic mice. The present study investigated whether young mice (4 months-old) with increased mitochondrial NOX4 levels recapitulate vascular aging and age-associated aortic stiffness. We generated transgenic mice with low (Nox4TG605; 2.1-fold higher) and high (Nox4TG618; 4.9-fold higher) mitochondrial NOX4 expression. Young Nox4TG618 mice showed significant increase in aortic stiffness and decrease in phenylephrine-induced aortic contraction, but not Nox4TG605 mice. Increased mitochondrial oxidative stress increased intrinsic VSMC stiffness, induced aortic extracellular matrix remodeling and fibrosis, a leftward shift in stress-strain curves, decreased volume compliance and focal adhesion turnover in Nox4TG618 mice. Nox4TG618 VSMCs phenocopied other features of vascular aging such as increased DNA damage, increased premature and replicative senescence and apoptosis, increased proinflammatory protein expression and decreased respiration. Aortic stiffening in young Nox4TG618 mice was significantly blunted with mitochondrial-targeted catalase overexpression. This demonstration of the role of mitochondrial oxidative stress in aortic stiffness will galvanize search for new mitochondrial-targeted therapeutics for treatment of age-associated vascular dysfunction.

Regulation of Vascular Smooth Muscle Cell Stiffness and Adhesion by [Ca2+]i: An Atomic Force Microscopy-Based Study - CORRIGENDUM.

The intracellular concentration of calcium ion ([Ca2+]i) is a critical regulator of cell signaling and contractility of vascular smooth muscle cells (VSMCs). In this study, we employed an atomic force microscopy (AFM) nanoindentation-based approach to investigate the role of [Ca2+]i in regulating the cortical elasticity of rat cremaster VSMCs and the ability of rat VSMCs to adhere to fibronectin (Fn) matrix. Elevation of [Ca2+]i by ionomycin treatment increased rat VSMC stiffness and cell adhesion to Fn-biofunctionalized AFM probes, whereas attenuation of [Ca2+]i by 1,2-Bis (2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis (acetoxymethyl ester) (BAPTA-AM) treatment decreased the mechanical and matrix adhesive properties of VSMCs. Furthermore, we found that ionomycin/BAPTA-AM treatments altered expression of α 5 integrin subunits and α smooth muscle actin in rat VSMCs. These data suggest that [Ca2+]i regulates VSMC elasticity and adhesion to the extracellular matrix by a potential mechanism involving changing dynamics of the integrin-actin cytoskeleton axis.