Primary Human Vascular Smooth Muscle Cells Research Articles

A pseudotyped adenovirus serotype 5 vector with serotype 49 fiber knob is an effective vector for vaccine and gene therapy applications

Adenoviruses (Ad) have demonstrated significant success as replication-deficient (RD) viral vectored vaccines, as well as broad potential across gene therapy and cancer therapy. Ad vectors transduce human cells via direct interactions between the viral fiber knob and cell surface receptors, with secondary cellular integrin interactions. Ad receptor usage is diverse across the extensive phylogeny. Commonly studied human Ad serotype 5 (Ad5), and chimpanzee Ad-derived vector “ChAdOx1” in licensed ChAdOx1 nCoV-19 vaccine, both form primary interactions with the Coxsackie and Adenovirus Receptor (CAR), which is expressed on human epithelial cells and erythrocytes. CAR usage is suboptimal for targeted gene delivery to cells with low/negative CAR expression, including human dendritic cells (DCs) and vascular smooth muscle cells (VSMCs). We evaluated the performance of a RD Ad5 vector pseudotyped with the fiber knob of human Ad serotype 49, termed Ad5/49K vector. Ad5/49K demonstrated superior transduction of murine and human DCs over Ad5, which translated into significantly increased T cell immunogenicity when evaluated in a mouse cancer vaccine model using 5T4 tumour-associated antigen. Additionally, Ad5/49K exhibited enhanced transduction of primary human VSMCs. These data highlight the potential of Ad5/49K vector for both vascular gene therapy applications and as a potent vaccine vector.

Abstract 2079: Single-cell And Spatially Resolved Transcriptome Analysis Reveals A Novel Lncrna Regulator Of Human Atherosclerosis

Cardiovascular diseases, such as atherosclerosis, are the major cause of death in Western societies. Still, molecular mechanisms of plaque destabilization remain unclear. Vascular Smooth Muscle Cells (VSMCs) are the most abundant cell type in atherosclerotic lesions. These cells keep their plasticity also with disease progression and are, therefore, an ideal target cell type for potential therapies. Long non-coding RNAs (lncRNAs) are one example of highly cell type- and disease-state-specific novel molecular modulators. We utilized combined total (bulk) RNA and single cell (sc) RNA sequencing of advanced human carotid artery lesions (patients who underwent carotid endarterectomy in our vascular surgery clinic) to discover novel regulators. Additionally, we performed hybridization-based RNA in situ sequencing (HybRISS, Xenium) to indicate where these genes are located within the plaques. We also performed scSeq in our inducible carotid artery plaque rupture mice ( ApoE-/- ). In vivo and in vitro modulation (KD; knock-down) of the newly identified lncRNA DLX6-AS1 ( D istal- L ess Homeobo x 6 A nti s ense 1 ) using Antisense LNA (Locked nucleic acid) GapmeRs is performed in this mouse model as well as in primary human VSMC culture. DLX6-AS1 shows a highly specific expression pattern in a distinct sub-cluster of VSMCs, as well as a striking location within human atherosclerotic lesions shown by spatial transcriptomics in advanced plaques. DLX6-AS1 is mainly expressed in the nucleus of cells, and KD of this lncRNA leads to changes in VSMC behavior in vitro and in vivo . The KD also has only little effect on the other genes of this locus, DLX6 and DLX5 ( D istal- L ess Homeobo x 6 and 5 ) We found Dlx6os1 ( D istal- l ess homeobo x 6, o pposite s trand 1 ) in our scRNA-seq dataset of the inducible carotid artery plaque rupture mice ( ApoE-/- ) where the KD leads to an increased plaque rupture rate. Taken together, our datasets, methods, and animal models demonstrate that utilizing bulk- and scRNA-seq data, together with spatially resolved sequencing methods, are powerful tools to identify and characterize novel lncRNA regulators that can facilitate fate decisions in VSMCs in atherosclerosis. DLX6-AS1 is one of these novel regulators and will be further analyzed in vivo and in vitro .

Sox9 Accelerates Vascular Aging by Regulating Extracellular Matrix Composition and Stiffness.

Vascular calcification and increased extracellular matrix (ECM) stiffness are hallmarks of vascular aging. Sox9 (SRY-box transcription factor 9) has been implicated in vascular smooth muscle cell (VSMC) osteo/chondrogenic conversion; however, its relationship with aging and calcification has not been studied. Immunohistochemistry was performed on human aortic samples from young and aged patients. Young and senescent primary human VSMCs were induced to produce ECM, and Sox9 expression was manipulated using adenoviral overexpression and depletion. ECM properties were characterized using atomic force microscopy and proteomics, and VSMC phenotype on hydrogels and the ECM were examined using confocal microscopy. In vivo, Sox9 was not spatially associated with vascular calcification but correlated with the senescence marker p16 (cyclin-dependent kinase inhibitor 2A). In vitro Sox9 showed mechanosensitive responses with increased expression and nuclear translocation in senescent cells and on stiff matrices. Sox9 was found to regulate ECM stiffness and organization by orchestrating changes in collagen (Col) expression and reducing VSMC contractility, leading to the formation of an ECM that mirrored that of senescent cells. These ECM changes promoted phenotypic modulation of VSMCs, whereby senescent cells plated on ECM synthesized from cells depleted of Sox9 returned to a proliferative state, while proliferating cells on a matrix produced by Sox9 expressing cells showed reduced proliferation and increased DNA damage, reiterating features of senescent cells. LH3 (procollagen-lysine, 2-oxoglutarate 5-dioxygenase 3) was identified as an Sox9 target and key regulator of ECM stiffness. LH3 is packaged into extracellular vesicles and Sox9 promotes extracellular vesicle secretion, leading to increased LH3 deposition within the ECM. These findings highlight the crucial role of ECM structure and composition in regulating VSMC phenotype. We identify a positive feedback cycle, whereby cellular senescence and increased ECM stiffening promote Sox9 expression, which, in turn, drives further ECM modifications to further accelerate stiffening and senescence.

In vitro Evaluation of the Calcification Inhibitory Properties of Policosanol, Genistein, and Vitamin D (Reduplaxin®) either Alone or in Combination

Introduction: The process of vascular calcification has severe clinical consequences in a number of diseases, including diabetes, atherosclerosis, and end-stage renal disease. In the present study, we investigated the effect of policosanol (Poli), genistein (Gen), and vitamin D (VitD) separately and in association to evaluate the possible synergistic action on inorganic phosphate (Pi)-induced calcification of vascular smooth muscle cells (VSMCs). Methods: Primary human VSMCs were cultured with either growth medium or growth medium supplemented with calcium and phosphorus (calcification medium) in combination with Poli, Gen, and VitD. Alizarin Red staining, mineralization, and the protein expression of RUNX2 and superoxide dismutase-2 (SOD2) were investigated. Results: All three substances tested were effective at reducing osteogenic differentiation of VSMCs in a dose-dependent manner. Poli+Gen, Poli+VitD, Gen+VitD treatment induced a greater inhibition of calcification and RUNX2 expression compared to single compounds treatments. Moreover, the association of Poli+Gen+VitD (Reduplaxin®) was more effective at inhibiting VSMCs mineralization and preventing the increase in RUNX2 expression induced by calcification medium but not modified SOD2 expression. Conclusions: The association of Pol, Gen, and VitD (Reduplaxin®) has an additive inhibitory effect on the calcification process of VSMCs induced in vitro by a pro-calcifying medium.

Effect of Mechanical Stimuli on the Phenotypic Plasticity of Induced Pluripotent Stem-Cell-Derived Vascular Smooth Muscle Cells in a 3D Hydrogel.

Introduction: Vascular smooth muscle cells (VSMCs) play a pivotal role in vascular homeostasis, with dysregulation leading to vascular complications. Human-induced pluripotent stem-cell (hiPSC)-derived VSMCs offer prospects for personalized disease modeling and regenerative strategies. Current research lacks comparative studies on the impact of three-dimensional (3D) substrate properties under cyclic strain on phenotypic adaptation in hiPSC-derived VSMCs. Here, we aim to investigate the impact of intrinsic substrate properties, such as the hydrogel's elastic modulus and cross-linking density in a 3D static and dynamic environment, on the phenotypical adaptation of human mural cells derived from hiPSC-derived organoids (ODMCs), compared to aortic VSMCs. Methods and results: ODMCs were cultured in two-dimensional (2D) conditions with synthetic or contractile differentiation medium or in 3D Gelatin Methacryloyl (GelMa) substrates with varying degrees of functionalization and percentages to modulate Young's modulus and cross-linking density. Cells in 3D substrates were exposed to cyclic, unidirectional strain. Phenotype characterization was conducted using specific markers through immunofluorescence and gene expression analysis. Under static 2D culture, ODMCs derived from hiPSCs exhibited a VSMC phenotype, expressing key mural markers, and demonstrated a level of phenotypic plasticity similar to primary human VSMCs. In static 3D culture, a substrate with a higher Young's modulus and cross-linking density promoted a contractile phenotype in ODMCs and VSMCs. Dynamic stimulation in the 3D substrate promoted a switch toward a contractile phenotype in both cell types. Conclusion: Our study demonstrates phenotypic plasticity of human ODMCs in response to 2D biological and 3D mechanical stimuli that equals that of primary human VSMCs. These findings may contribute to the advancement of tailored approaches for vascular disease modeling and regenerative strategies.

The effect of antibody-mediated PCSK9 inhibitor on bare-metal stent implantation outcome

Abstract Introduction Percutaneous coronary intervention (PCI) is the gold standard for the management of acute coronary syndrome, and has been benefited from advances in pharmacotherapy and device innovation. Nevertheless, in-stent restenosis and stent thrombosis remain serious complications following PCI procedure with stent implantation. Proprotein convertase subtilisin kexin type 9 (PCSK9) is an enzyme known for its role in plasma cholesterol homeostasis. Efficiently reducing plasma cholesterol, PCSK9 inhibition recently became a pivotal treatment strategy for hypercholesterolemia. However, the effect of PCSK9 inhibition on stent implantation outcomes remains uninvestigated. Methods Carotid artery bare-metal stent (BMS) implantation was performed on C57Bl/6 mice, which then a parallel assessment of various inflammatory mediators was performed. To inhibit PCSK9, alirocumab was administered weekly to antiplatelet-treated mice started before stent implantation. Six weeks after the stent implantation procedure, animals were harvested and histomorphometric analysis was performed on stented arteries. In vitro cell migration and cellular senescence were also assessed on alirocumab-treated primary human aortic endothelial (EC) and vascular smooth muscle cells (VSMC). Results Compared to sham intervention, stent implantation was associated with increased expression of several inflammatory mediators, including PCSK9. Interestingly, the increase in PCSK9 level was confirmed in the stented vascular tissue, but not in plasma (Fig 1A). Following alirocumab treatment, PCSK9 inhibition results in an increased intimal hyperplasia in the stented vascular segment of alirocumab-treated animals compared to controls (Fig 1B). In vitro, alirocumab promoted migration and inhibited the onset of senescence in primary human VSMC, while blunted the migration and increased the senescence of EC. Conclusion The results of this study demonstrated that antibody-based PCSK9 inhibition worsen bare-metal stent implantation outcome in mice. This observed effect is mediated, in part, by the differential effect on VSMC and EC senescence, thus promoting in-stent intimal hyperplasia while blunting vascular healing. The herein-reported data warrant additional investigations concerning the use of PCSK9 inhibitors in patients undergoing PCI with stent implantation.

Semaglutide treatment attenuates vessel remodelling in ApoE−/− mice following vascular injury and blood flow perturbation

Background and aimsRandomized clinical studies have shown a reduction in cardiovascular outcomes with glucagon-like peptide 1 receptor agonist (GLP-1RA) treatment with the hypothesized mechanisms being an underlying effect on atherosclerosis. Here, we aimed to assess the pharmacological effects of semaglutide in an atheroprone murine model that recapitulates central mechanisms related to vascular smooth muscle cell (VSMC) phenotypic switching and endothelial dysfunction known to operate within the atherosclerotic plaque. MethodsIn study A, we employed an electrical current to the carotid artery in ApoE−/− mice to induce severe VSMC injury and death, after which the arteries were allowed to heal for 4 weeks. In study B, a constrictive cuff was added for 6 h at the site of the healed segment to induce a disturbance in blood flow. ResultsCompared to vehicle, semaglutide treatment reduced the intimal and medial area by ∼66% (p = 0.007) and ∼11% (p = 0.0002), respectively. Following cuff placement, expression of the pro-inflammatory marker osteopontin and macrophage marker Mac-2 was reduced (p < 0.05) in the semaglutide-treated group compared to vehicle. GLP-1R were not expressed in murine carotid artery and human coronary vessels with and without atherosclerotic plaques, and semaglutide treatment did not affect proliferation of cultured primary human VSMCs. ConclusionsSemaglutide treatment reduced vessel remodelling following electrical injury and blood flow perturbation in an atheroprone mouse model. This effect appears to be driven by anti-inflammatory and -proliferative mechanisms independent of GLP-1 receptor-mediated signalling in the resident vascular cells. This mechanism of action may be important for cardiovascular protection.

Abstract 373: Senescence Drives Changes In Vesicle Secretion And Extracellular Matrix Organisation To Enhance Vascular Amyloidosis

Background: Vascular amyloidosis, caused when peptide monomers aggregate into insoluble amyloid, is a prevalent age-associated pathology. Aortic medial amyloid (AMA) is the most common human amyloid and is composed of medin, a 50-amino acid fragment of milk fat globule EGF-factor 8 (MFGE8). Emerging evidence has implicated extracellular vesicles (EVs) as mediators of pathological amyloid metabolism and accumulation in the extracellular matrix (ECM). MFGE8 has been identified as EV cargo in several cell types, including vascular smooth muscle cells (VSMCs). To determine the mechanisms of AMA formation with age, we explored the impact of VSMC senescence, EV secretion and ECM remodelling on medin accumulation. Methods: Primary human VSMCs were used for in vitro experiments, including EV isolation and synthesis of decellularised ECM for Western blotting, immunofluorescence and mass spectrometry analysis. Results: Medin was detected in EVs secreted from VSMCs. Small, round medin aggregates colocalised with EV markers in the ECM and medin was shown on the surface of EVs deposited in the ECM. Decreasing VSMC EV secretion with an inhibitor attenuated aggregation and deposition of medin in the ECM. Medin accumulation in the aortic wall of human subjects was strongly correlated with age. VSMC senescence increased EV secretion, increased EV medin loading and triggered extracellular deposition of medin in fibrillar form. Proteomic analysis showed VSMC senescence induced changes in EV cargo and ECM composition. These changes led to enhanced EV-ECM binding in the senescent ECM and accelerated aggregation of medin into amyloid fibrils. Abundance of the proteoglycan, HSPG2, was increased in the senescent ECM and colocalised with EVs and medin. Knock-down of HSPG2 decreased medin fibril formation in the senescent ECM. Conclusions: Medin is secreted into the ECM by VSMC-derived EVs, which enhance its aggregation. Senescence induces changes in EV secretion and cargo and ECM composition. Together, these changes trigger accumulation of fibrillar medin, contributing to the age-associated development of AMA.

Therapeutic MK2 inhibition blocks pathological vascular smooth muscle cell phenotype switch.

Vascular procedures, such as stenting, angioplasty, and bypass grafting, often fail due to intimal hyperplasia (IH), wherein contractile vascular smooth muscle cells (VSMCs) dedifferentiate to synthetic VSMCs, which are highly proliferative, migratory, and fibrotic. Previous studies suggest MAPK-activated protein kinase 2 (MK2) inhibition may limit VSMC proliferation and IH, although the molecular mechanism underlying the observation remains unclear. We demonstrated here that MK2 inhibition blocked the molecular program of contractile to synthetic dedifferentiation and mitigated IH development. Molecular markers of the VSMC contractile phenotype were sustained over time in culture in rat primary VSMCs treated with potent, long-lasting MK2 inhibitory peptide nanopolyplexes (MK2i-NPs), a result supported in human saphenous vein specimens cultured ex vivo. RNA-Seq of MK2i-NP–treated primary human VSMCs revealed programmatic switching toward a contractile VSMC gene expression profile, increasing expression of antiinflammatory and contractile-associated genes while lowering expression of proinflammatory, promigratory, and synthetic phenotype–associated genes. Finally, these results were confirmed using an in vivo rabbit vein graft model where brief, intraoperative treatment with MK2i-NPs decreased IH and synthetic phenotype markers while preserving contractile proteins. These results support further development of MK2i-NPs as a therapy for blocking VSMC phenotype switch and IH associated with cardiovascular procedures.

GFOGER Peptide Modifies the Protein Content of Extracellular Vesicles and Inhibits Vascular Calcification.

ObjectiveVascular calcification (VC) is an active process during which vascular smooth muscle cells (VSMCs) undergo an osteogenic switch and release extracellular vesicles (EVs). In turn, the EVs serve as calcification foci via interaction with type 1 collagen (COL1). We recently showed that a specific, six-amino-acid repeat (GFOGER) in the sequence of COL1 was involved in the latter’s interaction with integrins expressed on EVs. Our main objective was to test the GFOGER ability to inhibit VC.ApproachWe synthesized the GFOGER peptide and tested its ability to inhibit the inorganic phosphate (Pi)-induced calcification of VSMCs and aortic rings. Using mass spectrometry, we studied GFOGER’s effect on the protein composition of EVs released from Pi-treated VSMCs.ResultsCalcification of mouse VSMCs (MOVAS-1 cells), primary human VSMCs, and rat aortic rings was lower in the presence of GFOGER than with Pi alone (with relative decreases of 66, 58, and 91%, respectively; p < 0.001 for all) (no effect was observed with the scramble peptide GOERFG). A comparative proteomic analysis of EVs released from MOVAS-1 cells in the presence or absence of Pi highlighted significant differences in EVs’ protein content. Interestingly, the expression of some of the EVs’ proteins involved in the calcification process (such as osteogenic markers, TANK-binding kinase 1, and casein kinase II) was diminished in the presence of GFOGER peptide (data are available via ProteomeXchange with identifier PXD018169∗). The decrease of osteogenic marker expression observed in the presence of GFOGER was confirmed by q-RT-PCR analysis.ConclusionGFOGER peptide reduces vascular calcification by modifying the protein content of the subsequently released EVs, in particular by decreasing osteogenicswitching in VSMCs.

Prevention of aortic dissection and aneurysm via an ALDH2-mediated switch in vascular smooth muscle cell phenotype.

Aortic aneurysm/dissection (AAD) is a life-threatening disorder lacking effective pharmacotherapeutic remedies. Aldehyde dehydrogenase 2 (ALDH2) polymorphism is tied with various risk factors for AAD including hypertension, atherosclerosis, and hypercholesterolaemia although direct correlation between the two remains elusive. Two independent case-control studies were conducted involving 307 AAD patients and 399 healthy controls in two geographically distinct areas in China. Our data revealed that subjects carrying mutant ALDH2 gene possessed a ∼50% reduced risk of AAD compared with wild-type (WT) alleles. Using 3-aminopropionitrile fumarate (BAPN)- and angiotensin II (Ang II)-induced AAD animal models, inhibition of ALDH2 was found to retard development of AAD. Mechanistically, ALDH2 inhibition ablated pathological vascular smooth muscle cell (VSMC) phenotypical switch through interaction with myocardin, a determinant of VSMC contractile phenotype. Using microarray and bioinformatics analyses, ALDH2 deficiency was found to down-regulate miR-31-5p, which further altered myocardin mRNA level. Gain-of-function and loss-of-function studies verified that miR-31-5p significantly repressed myocardin level and aggravated pathological VSMC phenotypical switch and AAD, an effect that was blunted by ALDH2 inhibition. We next noted that ALDH2 deficiency increased Max expression and decreased miR-31-5p level. Moreover, ALDH2 mutation or inhibition down-regulated levels of miR-31-5p while promoting myocardin downstream contractile genes in the face of Ang II in primary human VSMCs. ALDH2 deficiency is associated with a lower risk of AAD in patients and mice, possibly via suppressing VSMC phenotypical switch in a miR-31-5p-myocardin-dependent manner. These findings favour a role for ALDH2 and miR-31-5p as novel targets for AAD therapy.

Interleukin-1β-Induced Senescence Promotes Osteoblastic Transition of Vascular Smooth Muscle Cells

Introduction: Interleukin (IL)-1β, as a key biomarker and mediator of vascular calcification in patients with end-stage renal disease (ESRD), may be involved in the process of premature senescence of vascular smooth muscle cells (VSMCs). This work sought to investigate whether IL-1β-induced premature senescence contributes to the process of osteoblastic transition and vascular calcification in VSMCs. Methods: Eighty-eight patients with ESRD (aged 25–81 years), 11 healthy individuals, and 15 cases of lesion-free distal radial arteries from dialysis ESRD patients with angiostomy were collected in this study. Immunohistochemical analysis was performed to detect expression of IL-1β, p21, and bone morphogenetic protein-2 (BMP2) in the distal radial arteries. Primary human VSMCs from healthy neonatal umbilical cords were incubated with test agents for 1–3 days. Intracellular levels of reactive oxygen species (ROS) and senescence-associated-β-galactosidase (SA-β-gal) staining were used to detect senescent cells. Alizarin red staining and the calcium content of the cell layer were used to detect mineral deposition in VSMCs. Results: Coincident with positive staining of IL-1β, p21, and BMP2 in the lesion-free distal radial arteries, 66.67% patients showed mineral deposition. Serum IL-1β was 0.24 ± 0.57, 1.20 ± 2.95, and 9.41 ± 40.52 pg/mL in 11 healthy individuals, 20 patients without calcification, and 53 patients with calcification, respectively. Analysis of the cross-table chi-square test showed cardiovascular calcification is not correlated with levels of serum IL-1β in patients with ESRD (p = 0.533). In response to IL-1β, VSMCs showed a senescence-like phenotype, such as flat and enlarged morphology, increased expression of p21, an increased activity of SA-β-gal, and increased levels of ROS. IL-1β-induced senescence of VSMCs was required for the activation of IL-1β/NF-κB/p53/p21 signaling pathway. IL-1β-induced senescent VSMCs underwent calcification due to osteoblastic transition mainly depending upon the upregulation of BMP2. Resveratrol, an activator of sirtuin-1, postponed the IL-1β-induced senescence through blocking the NF-κB/p53/p21 pathway and attenuated the osteoblastic transition and calcification in VSMCs. Conclusions: High levels of IL-1β in medial smooth muscles of arteries may play roles in inducing senescence-associated calcification. IL-1β-induced senescence depending on the activation of the NF-κB/p53/p21 signaling pathway and contributing to osteoblastic transition of VSMCs.

Transforming growth factor β1 suppresses proinflammatory gene program independent of its regulation on vascular smooth muscle differentiation and autophagy

Transforming growth factor β (TGFβ) signaling plays crucial roles in maintaining vascular integrity and homeostasis, and is established as a strong activator of vascular smooth muscle cell (VSMC) differentiation. Chronic inflammation is a hallmark of various vascular diseases. Although TGFβ signaling has been suggested to be protective against inflammatory aortic aneurysm progression, its exact effects on VSMC inflammatory process and the underlying mechanisms are not fully unraveled. Here we revealed that TGFβ1 suppressed the expression of a broad array of proinflammatory genes while potently induced the expression of contractile genes in cultured primary human coronary artery SMCs (HCASMCs). The regulation of TGFβ1 on VSMC contractile and proinflammatory gene programs appeared to occur in parallel and both processes were through a SMAD4-dependent canonical pathway. We also showed evidence that the suppression of TGFβ1 on VSMC proinflammatory genes was mediated, at least partially through the blockade of signal transducer and activator of transcription 3 (STAT3) and NF-κB pathways. Interestingly, our RNA-seq data also revealed that TGFβ1 suppressed gene expression of a battery of autophagy mediators, which was validated by western blot for the conversion of microtubule-associated protein light chain 3 (LC3) and by immunofluo-rescence staining for LC3 puncta. However, impairment of VSMC autophagy by ATG5 deletion failed to rescue TGFβ1 influence on both VSMC contractile and proinflammatory gene programs, suggesting that TGFβ1-regulated VSMC differentiation and inflammation are not attributed to TGFβ1 suppression on autophagy. In summary, our results demonstrated an important role of TGFβ signaling in suppressing proinflammatory gene program in cultured primary human VSMCs via the blockade on STAT3 and NF-κB pathway, therefore providing novel insights into the mechanisms underlying the protective role of TGFβ signaling in vascular diseases.

Abstract 323: Hydrogen Sulfide Limits the Development of Intimal Hyperplasia in a Mouse Model of Femoral Wire Injury and in Human Veins

Objectives: Mainstays of contemporary therapies for this arterial occlusive disease include angioplasties, stents, endarterectomies and bypass surgery. However, these treatments suffer from high failure rates due to re-occlusive vascular wall adaptations, namely intimal hyperplasia (IH). IH develops in response to endothelium injury, leading to inflammation, vascular smooth muscle cells (VSMC) dedifferentiation, migration and proliferation at the site of injury. Hydrogen sulfide (H 2 S) is a ubiquitous signaling gazotransmitter, which exhibits antioxidant, anti-inflammatory, and vaso-relaxant properties. Thus, we hypothesized that H 2 S could reduce IH formation. Methods: WT male C57BL6/J mice submitted to femoral wire injury surgery to induce IH were treated with an H 2 S donor (NaHS) in the drinking water. IH was measured 28 days post-surgery by histology. In addition, segments of great saphenous vein obtained from patients undergoing bypass surgery were maintained in culture ex-vivo for 7days in presence of various H 2 S donors (NaHS, GYY4137, diacyltrisulfide). Finally, primary human umbilical vein endothelial cells (HUVEC) and primary human VSMC were treated in-vitro with the same H 2 S donors to study cellular proliferation and migration. Results: NaHS treatment significantly reduced IH development in the mouse model of femoral wire injury ( Figure 1 ). Similarly, the various H 2 S donors prevented the development of IH in vein segments ex-vivo. In vitro , the same H 2 S donors stimulated human endothelial cells (HUVEC) migration and proliferation, while inhibiting migration and proliferation of primary VSMC ( Figure 2 ). Conclusions: Exogenous H 2 S prevents IH formation in mice in-vivo and in human veins ex-vivo . Importantly, H 2 S reduces VSMCs but stimulates ECs proliferation and migration. These data suggest that exogenous H 2 S therapy could be used in human to minimize IH, thus limiting vascular reconstruction failure.

Thrombomodulin Induces a Quiescent Phenotype and Inhibits Migration in Vascular Smooth Muscle Cells In Vitro

Loss of critical endothelial cell function and subsequent vascular smooth muscle cell (VSMC) migration is central to the pathology of injury-induced neointimal hyperplasia and recurrent stenosis. Thrombomodulin (TM), well known for its function as an endothelial surface anticoagulant, may have an unknown direct effect on VSMC physiology that would be lost after injury. Here, we examined a novel effect of TM on VSMC by testing the hypothesis that direct application of TM induces favorable changes to the morphology of VSMC and inhibits their migration. Primary human VSMC were harvested using the explant technique and used in early passage (1-4) for all experiments. Laser-scanning confocal fluorescent imaging was performed to assess the effect of soluble TM on VSMC morphology. In vitro, migration of VSMC was measured using: (1) a 4-hr modified Boyden chemotaxis assay and (2) a 24-hr electric cell-substrate impedance sensing injury migration assay. Migration experiments were conducted with VSMC exposed to increasing doses of soluble recombinant TM. Recombinant thrombin served as a positive control and serum-free media as a negative control for all experimentation. Data were analyzed using a Student's t-test or repeated measures analysis of variance where appropriate (α < 0.05). VSMC exposed to TM clearly demonstrated a quiescent morphology with organized stress fibers consistent with a quiescent, differentiated, contractile phenotype; whereas, thrombin stimulation led to an activated, dedifferentiated, synthetic phenotype. VSMC demonstrated a low, baseline level of migration in unstimulated serum-free conditions. Thrombin significantly stimulated VSMC migration as expected. TM, independent of thrombin, significantly inhibited baseline VSMC migration in a dose-response fashion. The maximal inhibition was observed at (5 μg/mL) with 70% reduction (56 ± 1.7 vs. 18 ± 3.5 cells/5 high-power fields, P = 0.0005). TM has a direct effect on VSMC resulting in a quiescent, differentiated and contractile phenotype, and inhibition of migration. This effect is independent of the presence of thrombin. These findings provide new knowledge in understanding the pathophysiology of vascular injury and support a strategy focused on restoring key endothelial function to prevent intimal hyperplasia.

Identification of the origin of circulating endothelial progenitor cells

Identification of the origin of circulating endothelial progenitor cells

Angiotensin-(1-7) and angiotensin-(1-9) inhibit vascular smooth muscle cell growth and migration in vitro and vascular remodelling in vivo

Background: Vascular smooth muscle cell(VSMC) proliferation and migration underlies the pathogenesis of atherosclerosis, vein graft failure and in-stent restenosis. Angiotensin II(AngII), acting via the AT1R, is integral in these processes. AngII is inhibited by the counter-regulatory axis of the renin angiotensin system, which is centered around the actions of angiotensin-converting-enzyme-2 and the production of Ang-(1-7) and Ang-(1-9), which act via the Mas receptor and AT2R, respectively. Here we investigated the role of Ang-(1-9) and Ang-(1-7) in primary human VSMC migration and proliferation, and in a mouse model of vascular injury. Methods: Migration was assessed via scratch assay and proliferation using the MTS-assay. Vascular injury was induced in vivo via wire injury to the left carotid artery. Ang-(1-7) and Ang-(1-9) were delivered via osmotic minipump and vascular remodeling quantified at 28 days post-injury. Results: VSMC migration and proliferation was inhibited by Ang-(1-9) and Ang-(1-7); these effects were selectively blocked by the pharmacological antagonists PD123,319 and A779, respectively, suggesting Ang-(1-9) acts via the AT2R and Ang-(1-7) via Mas. In vivo wire injury of the mouse carotid artery induced significant neointimal formation(NI) at 28 days post-injury; this was attenuated by Ang-(1-7) and Ang-(1-9) via Mas and the AT2R, respectively. Conclusion: These data demonstrate that Ang-(1-7) and Ang-(1-9) inhibit VSMC proliferation and migration in vitro and neointimal formation in vivo via the AT2R and Mas receptor, respectively. Here we demonstrate for the first time a direct biological effect of Ang-(1-9) within the vasculature. These findings highlight the potential of Ang-(1-9) and Ang-(1-7) as therapeutic agents in vascular injury.

YIA6 Medial Vascular Smooth Muscle Cell Cytopenia Accelerates Atherogenesis in APOE-/-MICE

Vascular smooth muscle cells (VSMC) are involved in many arterial diseases including atherosclerosis and aneurysm formation. VSMC accumulation and apoptosis have implications for atherosclerotic plaque stability and development. In a mouse model of inducible VSMC-specific apoptosis, chronic apoptosis of VSMC has been shown to induce features of plaque instability including fibrous cap thinning, plaque calcification, necrotic core enlargement, medial erosion, elastin breaks and accelerated atherogenesis of established atherosclerotic plaques. Based on previous findings, we questioned the role of medial VSMC in the development of atherosclerotic lesions. To test our research question, ApoE -/- and SM22α-hDTR/ApoE -/- mice were injected with 1 ng diphtheria toxin (DT) /gram bodyweight for 3 weeks to induce medial VSMC cytopenia. After 3 weeks, DT treatment was arrested, mice started a Western type diet (0.25% cholesterol) and were sacrificed after 6 and 18 weeks, respectively. The aortic arch including branches was excised and analysed (immune)histochemically. VSMC cytopenia after 3 weeks of DT treatment was verified by analysing vessel cellularity (cells/mm²) in both control (ApoE -/- ) and SM22α-hDTR/ApoE -/- mice. A 2-fold decrease in cellularity was observed in SM22α-hDTR/ApoE -/- mice as compared to the control. Atherosclerotic plaque size was quantified on HE stained sections. SM22α-hDTR/ApoE -/- mice displayed significantly accelerated plaque development as compared to control mice. In vitro , induction of apoptosis by ABT737 significantly induced calcification of primary human VSMC whereas ZVAD significantly inhibited calcification. Additional, conditioned medium from both apoptotic and calcified VSMC cultures served as a chemoattractant for macrophages. In vivo , these in vitro findings may explain the pro-inflammatory phenotype of atherosclerotic plaques. In conclusion, medial VSMC cytopenia results in accelerated development of established atherosclerotic plaques, with increased plaque calcification and vessel stenosis.

Panax notoginseng Saponins Attenuate Phenotype Switching of Vascular Smooth Muscle Cells Induced by Notch3 Silencing.

Panax notoginseng saponins (PNS) could maintain vascular smooth muscle cells (VSMCs) in stable phenotypes so as to keep blood vessel elasticity as well as prevent failing in endovascular treatment with stent. Downregulation of Notch3 expression in VSMCs could influence the phenotype of VSMCs under pathologic status. However, whether PNS is able to attenuate the Notch3 silencing induced phenotype switching of VSMCs remains poorly understood. Primary human VSMCs were transfected with a plasmid containing a small interfering RNA (siRNA) against Notch3 and then exposed to different doses of PNS. The control groups included cells not receiving any treatment and cells transfected with a control siRNA. Phenotypic switching was evaluated by observing cell morphology with confocal microscopy, as well as examining α-SM-actin, SM22α, and OPN using Western blot. Downregulated Notch3 with a siRNA induced apparent phenotype switching, as reflected by morphologic changes, decreased expression of α-SM-actin and SM22α and increased expression of OPN. These changes were inhibited by PNS in a dose-dependent manner. The phenotype switching of VSMCs induced by Notch3 knockdown could be inhibited by PNS in a dose-dependent manner. Our study provided new evidence for searching effective drug for amending stability of atherosclerotic disease.

Quercetin Attenuates Warfarin-induced Vascular Calcification in Vitro Independently from Matrix Gla Protein

Warfarin can stimulate vascular calcification in vitro via activation of β-catenin signaling and/or inhibition of matrix Gla protein (MGP) carboxylation. Calcification was induced in vascular smooth muscle cells (VSMCs) with therapeutic levels of warfarin in normal calcium and clinically acceptable phosphate levels. Although TGF/BMP and PKA pathways are activated in calcifying VSMCs, pharmacologic analysis reveals that their activation is not contributory. However, β-catenin activity is important because inhibition of β-catenin with shRNA or bioflavonoid quercetin prevents calcification in primary human VSMCs, rodent aortic rings, and rat A10 VSMC line. In the presence of quercetin, reactivation of β-catenin using the glycogen synthase kinase-3β (GSK-3β) inhibitor LiCl restores calcium accumulation, confirming that quercetin mechanism of action hinges on inhibition of the β-catenin pathway. Calcification in VSMCs induced by 10 μm warfarin does not associate with reduced levels of carboxylated MGP, and inhibitory effects of quercetin do not involve induction of MGP carboxylation. Further, down-regulation of MGP by shRNA does not alter the effect of quercetin. These results suggest a new β-catenin-targeting strategy to prevent vascular calcification induced by warfarin and identify quercetin as a potential therapeutic in this pathology.