Normal Vascular Smooth Muscle Cells Research Articles

Smooth Muscle Cofilin Confers Elasticity to Coronary Resistance Microvessels in Diabetes

Coronary resistance microvessels (CRMs) from type 2 diabetic (T2D) mice and pigs are less stiff compared to normal, a finding that is dictated by less stiff coronary vascular smooth muscle cells (VSMCs) Cofilin is an endogenous actin regulatory protein that depolymerizes filamentous F-actin, and recent studies have shown that portions of F-actin bound to cofilin are less stiff compared to their unbound F-actin counterparts. In this study, we tested the hypothesis that augmented cofilin confers elasticity to diabetic CRMs. CRMs were isolated and pooled (n=6 pooled CRMs per biological replicate) for proteomic data collection from both normal (n=3) and diabetic (n=5) mice. In separate experiments, we utilized deidentified normal and T2D human primary coronary VSMCs obtained from commercial vendors (Lonza and ATCC; n=4 per group) and experiments were performed on passage 5-8 VSMCs. Using these cells, we determined cofilin expression by Western blot, and cofilin siRNA (Cell Signaling Technology) was used 48 hours prior to experimentation to knock down cofilin expression. Elastic modulus, a measure of cell stiffness, was assessed by atomic force microscopy (AFM). Globular G-actin was labeled with fluorescein-deoxyribonuclease I (DNAse I) and F-actin was labeled with phalloidin. Images were taken in blinded manner and Image J was used to assess expression. In the proteomics screen, we observed zero cofilin spectral hits in the normal CRMs and 3-4 spectral hits in db/db CRMs (0.00 ± 0.0 vs. 3.40 ± 0.25, p<0.0001). Cofilin protein was increased in the coronary VSMCs of diabetic patients compared to normal coronary VSMCs (1.00 ± 0.16 vs. 1.837± 0.35, p=0.052). In human coronary VSMCs, silencing cofilin caused a significant increase in the F/G actin ratio when normal and diabetic data were pooled (scramble 3.029 ± 0.49 vs. siRNA 4.029 ± 0.68, p= 0.018, n=8). This increased F/G ratio was significant in the diabetic coronary VSMCs (scramble 2.636 ± 0.46 vs. siRNA 3.685 ± 0.41, p=0.049, n=4), and trended in the normal cells but did not reach statistical significance (scramble 3.423 ± 0.89 vs. cofilin siRNA 4.373 ± 1.37, p=0.224, n=4). Cofilin knockdown also caused a significant increase in elastic modulus by AFM when normal and diabetic data were pooled (scramble siRNA 1.814 ± 0.25 vs. cofilin siRNA 3.074 ± 0.35, p=0.0057, n=8). This trend persisted in the normal VMSCs (scramble siRNA 2.038 ± 0.31 vs. cofilin siRNA 3.445± 0.30, p=0.075, n=4) and in the diabetic VSMCs (scramble siRNA 1.589 ± 0.40 vs. cofilin siRNA 2.702 ± 0.62, p=0.085, n=4) but did not reach significance due to low available sample number. Overall, we demonstrate increased cofilin in human diabetic coronary VSMCs and in CRMs from diabetic mice, and silencing cofilin increased F/G actin ratio and increased cellular stiffness. These data support the notion that increased cofilin in T2D VSMCs may be the cause of decreased diabetic CRM stiffness. Support: NIH R00 HL116769, R01 HL165124, and S10 OD023438 and Nationwide Children’s Hospital to AJT. 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.

Autophagy reduces aortic calcification in diabetic mice by reducing matrix vesicle body-mediated IL-1β release

Vascular calcification (VC) is a common pathological process of cardiovascular disease that occurs in patients with type 2 diabetes mellitus (T2DM). However, the molecular basis of VC progression remains unknown. A GEO dataset (GSE146638) was analyzed to show that microbodies and IL-1β may play important roles in the pathophysiology of VC. The release of matrix vesicle bodies (MVBs) and IL-1β and the colocalization of IL-1β with MVBs or autophagosomes were studied by immunofluorescence in an in vivo diabetes mouse model with aortic calcification and an in vitro high glucose cell calcification model. MVB numbers, IL-1β levels and autophagy were increased in calcified mouse aortas and calcified vascular smooth muscle cells (VSMCs). IL-1β colocalized with MVBs and autophagosomes. The MVBs from calcified VSMCs induced the calcification of normal recipient VSMCs, and this effect was alleviated by silencing IL-1β. The autophagy inducer rapamycin reduced IL-1β expression and calcification in VSMCs, while these processes were induced by the autophagy inhibitor chloroquine. In conclusion, our results suggested that MVBs could carry IL-1β out of cells and induce VC in normal VSMCs, and these processes could be counteracted by autophagy. These results suggested that MVB-mediated IL-1β release may be an effective target for treating vascular calcification.

Mitochondrial Dysfunction and Increased DNA Damage in Vascular Smooth Muscle Cells of Abdominal Aortic Aneurysm (AAA-SMC).

There is increasing evidence for enhanced oxidative stress in the vascular wall of abdominal aortic aneurysms (AAA). Mitochondrial damage and dysfunction are hypothesized to be actors in altered production of reactive oxygen species (ROS) and oxidative stress. However, the role of mitochondria and oxidative stress in vascular remodelling and progression of AAA remains uncertain. We here addressed whether mitochondrial dysfunction is persistently increased in vascular smooth muscle cells (VSMCs) isolated from AAA compared to healthy VSMC. AAA-derived VSMC cultures (AAA-SMC, n = 10) and normal VSMC cultures derived from healthy donors (n = 7) were grown in vitro and analysed for four parameters, indicating mitochondrial dysfunction: (i) mitochondrial content and morphology, (ii) ROS production and antioxidative response, (iii) NADP+/NADPH content and ratio, and (iv) DNA damage, in the presence or absence of angiotensin II (AngII). AAA-SMC displayed increased mitochondrial circularity (rounded shape), reduced mitochondrial area, and reduced perimeter, indicating increased fragmentation and dysfunction compared to healthy controls. This was accompanied by significantly increased O2 - production, reduced NADP+/NADPH levels, a lower antioxidative response (indicated by antioxidative response element- (ARE-) driven luciferase reporter assays), more DNA damage (determined by percentage of γ-H2A.X-positive nuclei), and earlier growth arrest in AAA-SMC. Our data suggest that mitochondrial dysfunction and oxidative stress are persistently increased in AAA-SMC, emphasizing their implication in the pathophysiology of AAA.

Cell type-specific orientation and migration responses for a microgrooved surface with shallow grooves.

Directional cell migration due to mechanosensing for invivo microenvironment, such as microgrooved surfaces, is an essential process in tissue growth and repair in both normal and pathological states. Cell migration responses on the microgrooved surfaces might be reflected by the cell type difference, which is deeply involved in cellular physiological functions. Although the responses are implicated in focal adhesions (FAs) of cells, limited information is available about cell migration behavior on the microgrooved surfaces whose dimensions are comparable with the size of FAs. In the present study, we investigated the cell orientation and migration behavior of normal vascular smooth muscle cells (VSMCs) and cervical cancer HeLa cells on the microgrooved surface. The cells were cultured on the PDMS substrate comprising shallow grooves with 2-µm width and approximately 150-nm depth, which indicates the same order of magnitude as that of the horizontal and vertical size of FAs, respectively. The cell migration and intracellular structures were analyzed by live cell imaging and confocal fluorescence microscopy. The intracellular tension was also assessed using atomic force microscopy (AFM). VSMCs presenting well-aligned actin stress fibers with mature FAs revealed marked cell elongation and directional migration on the grooves; however, HeLa cells with nonoriented F-actin with smaller FAs did not. The internal force of the actin fibers was significantly higher in VSMCs than that in HeLa cells, and the increase or decrease in the cytoskeletal forces improved or diminished the sensing ability for shallow grooves, respectively. The results strongly indicated that directional cell migration should be modulated by cell type-specific cytoskeletal arrangements and intracellular traction forces. The differences in cell type-specific orientation and migration responses can be emphasized on the microgrooves as large as the horizontal and vertical size of FAs. The microgoove structure in the size range of the FA protein complex is a powerful tool to clarify subtle differences in the intracellular force-dependent substrate mechanosensing.

Role of nicotinamide phosphoribosyltransferase in delaying smooth muscle cell senescence and protecting abdominal aortic aneurysm

To investigate the protective effect of nicotinamide phosphoribosyltransferase (NAMPT) on abdominal aortic aneurysm by delaying the senescence of aortic vascular smooth muscle cells (VSMC). The primary VSMC cells from normal and patients with abdominal aortic aneurysm were cultured by tissue adherence method. Cells were divided into normal human-derived VSMC group (Ctrl-VSMC group), abdominal aortic aneurysm patient-derived VSMC group (AAA-VSMC group), and angiotensin II (Ang II) in vitro abdominal aortic aneurysm model group (Ang II-VSMC group, 100 nmol/L Ang II treated normal human-derived VSMC for 48 hours), Ang II + P7C3 group and AAA + P7C3 group after NAMPT agonist P7C3 intervention (adding 5 μmol/L P7C3 on the basis of Ang II-VSMC group and AAA-VSMC group, respectively). Immunofluorescence staining was used to identify VSMC; cell proliferation-associated antigen Ki67 staining was used to detect cell proliferation; senescence associated β-galactosidase (SA-β-gal) staining was used to detect cell senescence in each group; Western blotting was used to detect the protein expression levels of senescence-related proteins p21, p16 and NAMPT in each group. Compared with the Ctrl-VSMC group, the positive rate of SA-β-gal staining and the expression levels of senescence-related proteins p21 and p16 in the AAA-VSMC group and Ang II-VSMC group were significantly increased [SA-β-gal staining positive rate: (74.1±4.4)%, (68.6±5.5)% vs. (36.8±10.3)%, p21/GAPDH: 0.61±0.07, 0.51±0.03 vs. 0.31±0.03, p16/GAPDH: 0.77±0.03, 0.72±0.06 vs. 0.33±0.26, all P < 0.01]. However, the expression of NAMPT was significantly decreased (NAMPT/GAPDH: 0.88±0.07, 0.79±0.14 vs. 1.29±0.02, both P < 0.01). Compared with the Ang II-VSMC group, the positive rate of SA-β-gal staining and the expressions levels of senescence-related proteins p21 and p16 in the Ang II + P7C3 group were significantly lower [SA-β-gal staining positive rate: (49.1±3.2)% vs. (68.6±5.5)%, p21/GAPDH: 0.35±0.06 vs. 0.51±0.03, p16/GAPDH: 0.47±0.08 vs. 0.72±0.06, all P < 0.05], while the expression of NAMPT was significantly increased (NAMPT/GAPDH: 1.15±0.06 vs. 0.79±0.14, P < 0.01). Compared with the AAA-VSMC group, the positive rate of SA-β-gal staining and the expression levels of senescence-related proteins p21 and p16 in the AAA+P7C3 group were significantly lower [SA-β-gal staining positive rate: (54.1±6.0)% vs. (74.1±4.4)%, p21/GAPDH: 0.38±0.02 vs. 0.61±0.07, p16/GAPDH: 0.50±0.13 vs. 0.77±0.03, all P < 0.05], but the expression of NAMPT was significantly increased (NAMPT/GAPDH: 1.25±0.28 vs. 0.88±0.07, P < 0.01). NAMPT agonist P7C3 can delay the senescence of VSMC and play a protective role in abdominal aortic aneurysm.

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&lt;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.

Phenotypic and Functional Transformation in Smooth Muscle Cells Derived from a Superficial Thrombophlebitis-affected Vein Wall

Superficial thrombophlebitis (ST) is a frequent pathology, but its exact incidence remains to be determined. This study tested the hypothesis whether relationships exist among smooth muscle cells (SMCs) derived from ST, varicose great saphenous veins (VGSVs), and normal great saphenous veins (GSVs). Forty-one samples of ST, VGSVs, and GSVs were collected. SMCs were isolated and cultured. Proliferation, migration, adhesion, and senescence in SMCs from the three vein walls were compared by various methods. Bax, Bcl-2, caspase-3, matrix metalloproteinase-2 (MMP-2), MMP-9, tissue inhibitor of metalloproteinase-1 (TIMP-1), and TIMP-2 messenger RNA (mRNA) and protein expressions were detected by fluorescence quantitative PCR and Western blot. An obvious decrease in cytoskeletal filaments was observed in thrombophlebitic vascular smooth muscle cells (TVSMCs). The quantity of proliferation, migration, adhesion, and senescence in TVSMCs was significantly higher than in varicose vascular smooth muscle cells and normal vascular smooth muscle cells (NVSMCs) (all P < 0.05). Bax and caspase-3 mRNA and protein expression were decreased, while Bcl-2 mRNA and protein expression were increased in the TVSMCs compared with the varicose vascular smooth muscle cells and the NVSMCs (all P < 0.05). MMP-2, MMP-9, TIMP-1, and TIMP-2 mRNA and protein expression were significantly increased in the TVSMCs compared with the VVGSVs and the NVSMCs (all P < 0.05). SMCs derived from ST are more dedifferentiated and demonstrate increased cell proliferation, migration, adhesion, and senescence, as well as obviously decreased cytoskeletal filaments. These results suggest that the phenotypic and functional differences could be related to the presence of atrophic and hypertrophic vein segments during the disease course among SMCs derived from ST, VGSVs, and GSVs.

Regulation of reactive oxygen species in the pathogenesis of matrix vesicles induced calcification of recipient vascular smooth muscle cells.

Increased oxidative stress is associated with vascular calcification in patients with chronic kidney disease (CKD). We have previously demonstrated that cellular-derived matrix vesicles (MV), but not media-derived MV, are endocytosed in the presence of phosphorus by recipient normal rat vascular smooth muscle cells (VSMC) and induce calcification through ERK1/2 and [Ca2+]i signaling. We hypothesized that these changes were mediated by increased reactive oxygen species (ROS) production. MV were co-cultured with recipient VSMC in the presence of high phosphorus and ROS production and cell signaling assessed. The results demonstrated MV endocytosis led to increased ROS production in recipient VSMC with no increase in mitochondrial oxygen consumption or oxidative phosphorylation (OXPHOS), indicating the ROS was not from the mitochondria. The use of inhibitors demonstrated that endocytosis of these MV by VSMC led to a signaling cascade in the cytoplasm beginning with ERK1/2 signaling, then increased [Ca2+]i and stimulation of ROS production, mediated by nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX)1/4. Media-derived MV did not induce this cascade, indicating endocytosis itself was not a factor. Furthermore, inhibition of either ERK1/2 activation or [Ca2+]i reduced vascular calcification. We conclude that endocytosis of pro-mineralizing MV can induce a series of signaling events in normal VSMC that culminate in generation of ROS via activation of NOX1/4. Understanding these pathways will allow the development of future targeted therapeutics.

Activating BK channels ameliorates vascular smooth muscle calcification through Akt signaling.

Vascular calcification (VC) is characterized by pathological depositions of calcium and phosphate in the arteries and veins via an active cell-regulated process, in which vascular smooth muscle cells (VSMCs) transform into osteoblast/chondrocyte-like cells as in bone formation. VC is associated with significant morbidity and mortality in chronic kidney disease (CKD) and cardiovascular disease, but the underlying mechanisms remain unclear. In this study we investigated the role of large-conductance calcium-activated potassium (BK) channels in 3 experimental VC models. VC was induced in vascular smooth muscle cells (VSMCs) by β-glycerophosphate (β-GP), or in rats by subtotal nephrectomy, or in mice by high-dosage vitamin D3. We showed that the expression of BK channels in the artery of CKD rats with VC and in β-GP-treated VSMCs was significantly decreased, which was functionally confirmed by patch-clamp recording. In β-GP-treated VSMCs, BK channel opener NS1619 (20 μM) significantly alleviated VC by decreasing calcium content and alkaline phosphatase activity. Furthermore, NS1619 decreased mRNA expression of ostoegenic genes OCN and OPN, as well as Runx2 (a key transcription factor involved in preosteoblast to osteoblast differentiation), and increased the expression of α-SMA protein, whereas BK channel inhibitor paxilline (10 μM) caused the opposite effects. In primary cultured VSMCs from BK-/- mice, BK deficiency aggravated calcification as did BK channel inhibitor in normal VSMCs. Moreover, calcification was more severe in thoracic aorta rings of BK-/- mice than in those of wild-type littermates. Administration of BK channel activator BMS191011 (10 mg· kg-1 ·d-1) in high-dosage vitamin D3-treated mice significantly ameliorated calcification. Finally, co-treatment with Akt inhibitor MK2206 (1 μM) or FoxO1 inhibitor AS1842856 (3 μM) in calcified VSMCs abrogated the effects of BK channel opener NS1619. Taken together, activation of BK channels ameliorates VC via Akt/FoxO1 signaling pathways. Strategies to activate BK channels and/or enhance BK channel expression may offer therapeutic avenues to control VC.

Exosomes from vascular smooth muscle cells of diabetic origin promote a pro‐inflammatory macrophage phenotype

Diabetes Mellitus accounts for over 350,000 deaths annually, within the United States alone. One of the most common co-morbidities of diabetes is heart disease. Vascular inflammation among diabetic patients leads to a two-to-four fold increase in risk of heart attack and stroke. Two specific micro-RNAs (miRNA), miR-221 and miR-222, promote vascular smooth muscle cell (VSMC) proliferation and are elevated in VSMCs of diabetic subjects. Diabetic VSMC derived exosomes (DVEs) also exhibit elevated levels of miR-221 and miR-222 compared to exosomes isolated from normal VSMCs (NVEs), suggesting a novel paracrine signaling method mediating the cardiovascular complications of diabetes. The objective of this study is to determine whether this mechanism alters macrophage phenotype. Macrophages exist in multiple phenotypes, including a pro-inflammatory state (M1) and a resolving state (M2). We hypothesize that exposure of macrophages to DVEs will promote polarization toward the M1 phenotype. CD14+ human monocytes were differentiated to macrophages by a 6-day serum incubation. Macrophages were then co-incubated with DVEs or NVEs overnight. M1 versus M2 polarization was assessed using a score based on expression of seven RNA markers of macrophage phenotype. Additional studies were performed using DVEs and NVEs isolated from VSMCs following knockdown of miR-221/222. Exposure of macrophages to DVEs promoted a significant increase in M1 polarization. This polarization was diminished in the DVEs isolated from VSMCs following miR-221/222 knockdown. DVE exposure was also associated with diminished expression of an RNA-binding protein, quaking (QKI), a known target of miR-221/222 whose deficiency is associated with M1 polarization. We conclude that exosomal transfer of miR-221/222 from diabetic VSMCs to macrophages promotes M1 polarization through down-regulation of QKI. These findings suggest that this paracrine signaling mechanism represents a novel mechanism underlying the cardiovascular complications of diabetes.

Effect of MicroRNA-26a Targeting mCTGF on Calcification of Vascular Smooth Muscle Cells Through Regulating Osteoprotegerin/Receptor Activator of NF-kB/Receptor Activator of Nuclear Factor Kappa-B Ligand Signaling Pathway

To explore the effect of microRNA-26a targeting mCTGF on inhibiting the calcification of vascular smooth muscle cells (VSMCs) through OPG/RANK/RANKL signaling pathway. VSMCs were isolated and cultured by adherent method. The morphology of VSMCs was observed under microscope and the surface antigen was identified by flow cytometry. The calcification of VSMCs was detected using alizarin red S staining and oil red O staining. microRNA-26a level in supernatant of cultured VSMCs was detected by ultracentrifugation method. The binding site between microRNA-26a and 3′-UTR of mCTGF was predicted by bioinformatics software; VSMCs were induced to be calcified and verified by the luciferase reporter gene assay. The mRNA and protein expressions of OPG/RANK/RANKL signaling pathway were measured by qRT-PCR and Western blotting. Normal VSMCs were assigned into normal group (normal VSMCs were treated with the exosomes secreted by normal VSMCs), CKD group (normal VSMCs were treated with the exosomes secreted by VSMCs in mice with chronic kidney disease (CKD)) and CKD/overexpressed mCTGF group (normal VSMCs were treated with the exosomes secreted by VSMCs in CKD mice and meanwhile treated with overexpressed mCTGF) followed by analysis of OPG/RANK/RANKL signaling level by qRT-PCR and ALP activity. VSMCs grew against the wall and showed a long fusiform shape. Calcification and adipogenesis of VSMCs was found under different induction conditions. microRNA-26a level in the supernatant of VSMCs in CKD group was significantly higher than normal group; after microRNA-26a bound to of mCTGF, mCTGF level in the CKD/overexpressed mCTGF group was significantly lower. The expression of OPG/RANK/RANKL signaling pathway was significantly decreased in the CKD/overexpressed mCTGF group. Meanwhile, OPG/RANK/RANKL signaling protein was increased significantly after treatment of exosomes secreted by VSMCs in CKD mice. mCTGF is the target gene of microRNA-26a and microRNA-26a can target mCTGF to regulate exosomes secretion by VSMCs and OPG/RANK/RANKL signaling to inhibit the calcification of VSMCs.

Macroscopic and microscopic analysis of the mechanical properties and adhesion force of cells using a single cell tensile test and atomic force microscopy: Remarkable differences in cell types

Many experimental techniques have been reported to provide knowledge of the mechanical behavior of cells from biomechanical viewpoints, however, it is unclear how the intercellular structural differences influence macroscopic and microscopic mechanical properties of cells. The aim of our study is to clarify the comprehensive mechanical properties and cell-substrate adhesion strength of cells, and the correlation with intracellular structure in different cell types. We developed an originally designed micro tensile tester, and performed a single cell tensile test to estimate whole cell tensile stiffness and adhesion strength of normal vascular smooth muscle cells (VSMCs) and cervical cancer HeLa cells: one half side of the specimen cell was lifted up by a glass microneedle, then stretched until the cell detached from the substrate, while force was simultaneously measured. The tensile stiffness and adhesion strength were 49 ± 10 nN/% and 870 ± 430 nN, respectively, in VSMCs (mean ± SD, n = 8), and 19 ± 17 nN/% and 320 ± 160 nN, respectively, in HeLa cells (n = 9). The difference was more definite in the surface elastic modulus map obtained by atomic force microscopy, indicating that the internal tension of the actin cytoskeleton was significantly higher in VSMCs than in HeLa cells. Structural analysis with confocal microscopy revealed that VSMCs had a significant alignment of F-actin cytoskeleton with mature focal adhesion, contrary to the randomly oriented F-actin with smaller focal adhesion of HeLa cells, indicating that structural arrangement of the actin cytoskeleton and their mechanical tension generated the differences in cell mechanical properties and adhesion forces. The finding strongly suggests that the mechanical and structural differences in each cell type are deeply involved with their physiological functions.

Targeted Repair of Vascular Injury by Adipose-Derived Stem Cells Modified with P-Selectin Binding Peptide.

Percutaneous coronary intervention for coronary artery disease treatment often results in pathological vascular injury, characterized by P‐selectin overexpression. Adipose‐derived stem cells (ADSCs) therapeutic efficacy remains elusive due to poor ADSCs targeting and retention in injured vessels. Here, conjugated P‐selectin binding peptide (PBP) to polyethylene glycol‐conjugated phospholipid derivative (DMPE‐PEG) linkers (DMPE‐PEG‐PBP; DPP) are used to facilitate the modification of PBP onto ADSCs cell surfaces via hydrophobic interactions between DMPE‐PEG and the phospholipid bilayer. DPP modification neither has influence on ADSCs proliferation nor apoptosis/paracrine factor gene expression. A total of 5 × 10−6 m DPP‐modified ADSCs (DPP‐ADSCs) strongly binds to P‐selectin‐displaying activated platelets and endothelial cells (ECs) in vitro and to wire‐injured rat femoral arteries when administered by intra‐arterial injection. Targeted binding of ADSCs shields injury sites from platelet and leukocyte adhesion, thereby decreasing inflammation at injury sites. Furthermore, targeted binding of ADSCs recovers injured ECs functionality and reduces platelet‐initiated vascular smooth muscle cells (VSMCs) chemotactic migration. Targeted binding of DPP‐human ADSCs to balloon‐injured human femoral arteries is also demonstrated in ex vivo experiments. Overall, DPP‐ADSCs promote vascular repair, inhibit neointimal hyperplasia, increase endothelium functionality, and maintain normal VSMCs alignment, supporting preclinical noninvasive utilization of DPP‐ADSCs for vascular injury.

MicroRNA‐34c suppresses proliferation of vascular smooth muscle cell via modulating high mobility group box protein 1

BackgroundAtherosclerosis is the most frequent pathological process that causes cardiovascular diseases.ObjectiveThe present study aimed to confirm miRNAs associated with atherosclerosis and explore the molecular mechanism of miR‐34c and its target high mobility group box protein 1 (HMGB1) in the control of growth of smooth muscle cells in the development of atherosclerosis.MethodsReal‐time PCR was firstly performed to confirm miRNA correlation with atherosclerosis, and computational analysis and luciferase assay were performed to explore the target of miR‐34c, Western blot, and real‐time PCR were also utilized to reveal the effect of whether high glucose (HG) and miR‐34c affect miR‐34c, HMGB1 levels, NF‐κB p65 and TNF‐α levels, and the role of miR‐34c on vascular smooth muscle cells (VSMCs) viability induced by HG. Students' unpaired t test was performed to compare data between two groups.ResultsMiR‐34c level was associated with atherosclerosis with different expression between VSMCs treated with high glucose or normal VSMCs. Then, HMGB1 is a virtual target of miR‐34c with predicted binding site resided in HMGB1 3′UTR and further verified by that miR‐34c remarkably reduced luciferase activity of wild HMGB1 3′UTR under a concentration‐dependent fashion, and miR‐34c cannot influence luciferase activity of mutant HMGB1 3′UTR.ConclusionsThe results suggested miR‐34c might be a novel therapeutic strategy in the management of atherosclerosis by suppressing the expression of HGMB1 and its downstream effectors.

LncRNA KCNQ1OT1 Suppresses the Inflammation and Proliferation of Vascular Smooth Muscle Cells through IκBa in Intimal Hyperplasia

Inflammation and proliferation of vascular smooth muscle cells (VSMCs) are the key events in intimal hyperplasia. This study aimed to explore the mechanism by which long non-coding RNA (lncRNA) KCNQ1OT1 affects VSMC inflammation and proliferation in this context. A vein graft (VG) model was established in mice to introduce intimal hyperplasia. Isolated normal VSMCs were induced with platelet-derived growth factor type BB (PDGF-BB), and the cell proliferation, migration, and secretion of inflammatory factors were determined. The results showed that KCNQ1OT1 was downregulated in the VSMCs from mice with intimal hyperplasia and in the PDGF-BB-treated VSMCs, and such downregulation of KCNQ1OT1 resulted from the increased methylation level in the KCNQ1OT1 promoter. Overexpressing KCNQ1OT1 suppressed PDFG-BB-induced VSMC proliferation, migration, and secretion of inflammatory factors. In VSMCs, KCNQ1OT1 bound to the nuclear transcription factor kappa Ba (IκBa) protein and increased the cellular IκBa level by reducing phosphorylation and promoting ubiquitination of the IκBa protein. Meanwhile, KCNQ1OT1 promoted the expression of IκBa by sponging miR-221. The effects of KCNQ1OT1 knockdown on promoting VSMC proliferation, migration, and secretion of inflammatory factors were abolished by IκBa overexpression. The roles of KCNQ1OT1 in reducing the intimal area and inhibiting IκBa expression were proved in the VG mouse model after KCNQ1OT1 overexpression. In conclusion, KCNQ1OT1 attenuated intimal hyperplasia by suppressing the inflammation and proliferation of VSMCs, in which the mechanism upregulated IκBa expression by binding to the IκBa protein and sponging miR-221.

Abstract TP556: Role of Vascular Smooth Muscle Cells in Diabetes-related Vascular Cognitive Impairment

The incidence of vascular cognitive impairment is increased 2-3 folds in type 2 diabetes, but the underlying mechanisms have not been fully elucidated. The present study examines whether chronic hyperglycemia and associated enhanced ROS production damage cerebral vascular smooth muscle cells (VSMCs) that promote impairment of cerebral autoregulation and contribute to cognitive deficits. The levels of glucose (422 ± 32 vs. 94 ± 3 mg/dL) and glycated hemoglobin (HbA1c, 11.5 ± 0.2 vs. 4.3 ± 0.1%) were higher in old (12-month) T2DN (n=15) in comparison with SD control rats (n=6). We found that the actin cytoskeleton was disorganized in VSMCs freshly isolated from middle cerebral arteries (MCAs) of T2DN compared with SD rats, similar to what was seen in normal primary VSMCs treated with high glucose or H 2 O 2 . Superoxide production was elevated in VSMCs and MCAs freshly isolated from diabetic rats. Elderly diabetic rats exhibit impaired myogenic response of MCA. Cortical blood flow measured by laser Doppler flowmetry rose by 137 ± 15% vs. 36 ± 5% in diabetic (n=6) vs. normal rats (n=6) when MAP was increased from 100 to 180 mmHg. T2DN rats exhibited higher levels of IL-1 β and IL-2, and marked neurodegeneration in the hippocampus and cortex. Elderly T2DN rats showed learning and memory impairments using 8-arm water maze testing. These results indicate that hyperglycemia and elevated ROS production induce VSMCs dysfunction in diabetic rats, which contribute to cerebral vascular dysfunction and promote development of cognitive deficits.

10 - Electron transport chain inhibitors alter membrane potential and induce mitochondrial fragmentation in vascular smooth muscle cells: implications for bioenergetic measurements

Mitochondrial metabolism relies on membrane potential (ΔΨm) for effective electron transport across respiratory complexes and a filamentous mitochondrial network. Oxidative metabolism is typically measured via bioenergetic assays utilizing electron transport chain inhibitors including oligomycin (ATP synthase inhibitor), FCCP (proton ionophore), antimycin A (Complex III inhibitor) and rotenone (Complex I inhibitor). Bioenergetic effects of these compounds are well-established, but effects on ΔΨm and morphology in vascular smooth muscle cells (VSMC) remains largely unknown. Pulmonary hypertension (PH) is complex vasculopathy characterized by excess cell proliferation and a metabolic defect – PH VSMC preferentially use aerobic glycolysis for energy production, having experienced a metabolic switch similar to other proliferative diseases, including cancer. Using normal and PH patient pulmonary artery smooth muscle cells (hPASMC), mitochondrial metabolism was measured via micropolarimetry using oligomycin, FCCP, Antimycin A and rotenone. Mitochondrial morphology was assessed pre- and post-inhibitor treatment by confocal imaging of MitoTracker Deep Red-loaded hPASMC, and subsequent application of a machine learning algorithm to predict mitochondrial shape and calculate volume. ΔΨm was measured pre- and post- inhibitor treatment by flow cytometry of tetramethylrhodamine (TMRE)-loaded hPASMC. Compared to PH, normal hPASMC exhibit significantly higher baseline oxidative metabolism. PH PASMC display significant mitochondrial fragmentation (~60% intermediate/fragmented); normal PASMC are highly fused (~75% filamentous). ETC inhibitors induce fragmentation in normal PASMC, significantly reducing filamentous network in favor of intermediate rods. Fragmentation is further enhanced in ETC-inhibited PH PASMC (>80% fragmented/intermediate). ETC inhibitors also induce a 10-50% reduction of ΔΨm in normal PASMC. PH PASMC are comparatively hyperpolarized, and largely resistant to inhibitor-induced depolarization, suggesting the underlying metabolic defect in PH PASMC is independent of ΔΨm. ETC inhibitors used to study oxidative metabolism have off-target effects in normal VSMC, causing membrane depolarization and fragmentation. In PH patients, the mitochondrial network is destabilized and sensitive to further fragmentation by ETC inhibition, but this fragmentation is independent of membrane potential.

Arsenic trioxide induces growth inhibition and death in human pulmonary artery smooth muscle cells accompanied by mitochondrial O2•- increase and GSH depletion.

Arsenic trioxide (ATO; As2 O3 ) induces cell death in various cells via oxidative stress. Expose to chronic arsenic is involved in the development of vascular diseases. However, little is known about the cytotoxic effects of ATO on human normal vascular smooth muscle cells (VSMCs). Thus, in this study, we investigated the effects of ATO on cell growth and death in human pulmonary artery smooth muscle (HPASM) cells in relation to reactive oxygen species (ROS) and glutathione (GSH) levels. ATO treatment decreased the growth of HPASM cells with an IC50 of ∼30-50 μM at 24 h, and ATO induced HPASM cell death via apoptosis or necrosis dependent on the doses of it at this time. Treatment with 50 μM ATO did not increase ROS levels at the early time points, but it significantly increased mitochondrial O2•- levels at 24 h. ATO also induced GSH depletion in HPASM cells. N-acetyl cysteine (NAC; a well-known antioxidant) did not significantly affect apoptotic cell death, ROS levels, or GSH depletion in ATO-treated HPASM cells. However, l-buthionine sulfoximine (BSO; an inhibitor of GSH synthesis) intensified mitochondrial O2•- levels in ATO-treated HPASM cells, and significantly increased cell death and GSH depletion in these cells as well. In summary, we provided the first evidence that ATO inhibited the growth of HPASM cells, and induced apoptotic and/or necrotic cell death in these cells, accompanied by increases in mitochondrial O2•- level and GSH depletion.

Type 2 Diabetic Coronary Vascular Smooth Muscle Cells Exhibit Decreased Stiffness and Decreased Adhesion

We previously reported that type 2 diabetic (T2D) coronary resistance microvessels (CRMs) undergo early inward hypertrophic remodeling with reduced vessel stiffness that is associated with a proliferative VSMC phenotype. We also previously revealed that there is no difference in stiffness of decellularized CRMs from diabetic (db/db) and normal (Db/db) mice, suggesting that the decreased stiffness of the intact diabetic CRM is not due to alterations in the extracellular matrix (ECM) of those microvessels. Given that diabetic CRMs are less stiff and this decreased stiffness does not appear to be due differences in the mechanical strength of the surrounding ECM, the aim of the current study was to determine whether a reduction in cellular mechanical stiffness between Db/db and db/db CRM vascular smooth muscle cells (VSMCs) may be responsible. VSMCs were isolated from 16 week old Db/db and db/db mice and passage 1 cells were used for all experiments (n=6–7 for all groups). Using an atomic force microscope with probes coated with fibronectin (FN), a nano‐indentation protocol was used to measure cellular stiffness and adhesion to FN. Elastic modulus, a measurement of cellular stiffness, was reduced in diabetic coronary VSMCs compared to normal (Db/db: 6.74 ± 0.40 kPa vs. db/db: 4.96 ± 0.35 kPa, p = 0.004). By comparison, there were no significant differences in elastic modulus between normal and diabetic aortic VMSCs (Db/db: 5.39 ± 0.79 kPa vs. db/db: 5.18 ± 0.87 kPa, p = 0.861). Additionally, diabetic coronary VSMCs had reduced adhesive force to fibronectin compared to normal coronary VSMCs (Db/db: 54.78 ± 5.49 vs. db/db: 39.8 ± 3.43 pN, p = 0.03). In a separate experiment, qPCR was performed on mRNA from normal and diabetic CRM VSMCs. Alpha‐smooth muscle actin trended lower in db/db CRM VSMCs (p=0.07), indicating a de‐differentiated phenotype. Cell cycle regulator, p53, had significantly reduced mRNA expression in the diabetic coronary VSMCs over control VSMCs (Db/db 1.00±0.06 vs. db/db 0.63±0.08, p&lt;0.01). These data show that reductions in cellular stiffness and adhesion to the ECM of diabetic CRM VSMCs may underlie ex vivo whole microvessel mechanical properties to account for reduced CRM stiffness. The changes in diabetic coronary VSMC stiffness and adhesion may be the result of VMSC phenotypic switching to a more de‐differentiated/proliferative phenotype.Support or Funding InformationSupport: NIH R00 HL116769 and S10 OD023438 and Nationwide Children's Hospital to AJTThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Not all vascular smooth muscle cell exosomes calcify equally in chronic kidney disease

Prevention of medial calcification in patients with chronic kidney disease requires the maintenance of vascular smooth muscle cell fitness. To preserve viability under chronic kidney disease-induced stress, vascular smooth muscle cells increase exosome formation and release, but the result is aggravated pathological calcification. Now Chen etal. report that microvesicles from calcifying vascular smooth muscle cells may propagate procalcifying signals to normal vascular smooth muscle cells. To help design effective strategies to impair procalcifying cell-to-cell communication, this commentary updates the current understanding of the main regulators of microvesicle/exosome biogenesis and secretion.