Morphology Of Smooth Muscle Cells Research Articles

High salt-induced morphological and glycocalyx remodeling of human vascular smooth muscle cells is reversible but induces a high sodium salt-like sensitive memory.

Our recent work showed that short-term treatment (1-2 days) with high sodium salt had no effect on the morphology of human vascular smooth muscle cells (hVSMCs). However, chronic (long-term treatment, 6-16 days) high sodium salt (CHSS) induced hypertrophy and decreased the relative density of the glycocalyx in hVSMCs. Whether this CHSS effect is reversible at both the morphological and the intracellular calcium and sodium levels is unknown. In the present study, we tested the hypothesis that the effect of CHSS on the morphological and functional levels of hVSMCs is reversible. However, it induced an irreversible increase in the sensitivity of the cells following short-term treatment with high extracellular Na+. We tested the effects of the removal of CHSS treatment on the morphology and intracellular sodium and calcium of hVSMCs. Our results showed that restoring average sodium concentration (145 mM) modeled back the relative density of the glycocalyx, the intracellular resting calcium and sodium levels, and the whole cell and nuclear volumes of hVSMCs. In addition, it induced a permanent remodeling of hVSMCs' response to a short-term increase in the extracellular level of sodium salt by developing spontaneous cytosolic and nuclear calcium waves. Our results showed that CHSS is reversible at both the morphological and basal intracellular ionic levels. However, it maintained a high sensitivity to short-term elevation of extracellular sodium. These results suggest that even if chronic high salt is corrected, it induces a high sodium salt-like sensitive memory.

Corneal injury is associated with stromal and vascular alterations within cranial dura mater.

The cornea and cranial dura mater share sensory innervation. This link raises the possibility that pathological impulses mediated by corneal injury may be transmitted to the cranial dura, trigger dural perivascular/connective tissue nociceptor responses, and induce vascular and stromal alterations affecting dura mater blood and lymphatic vessel functionality. In this study, using a mouse model, we demonstrate for the first time that two weeks after the initial insult, alkaline injury to the cornea leads to remote pathological changes within the coronal suture area of the dura mater. Specifically, we detected significant pro-fibrotic changes in the dural stroma, as well as vascular remodeling characterized by alterations in vascular smooth muscle cell (VSMC) morphology, reduced blood vessel VSMC coverage, endothelial cell expression of the fibroblast specific protein 1, and significant increase in the number of podoplanin-positive lymphatic sprouts. Intriguingly, the deficiency of a major extracellular matrix component, small leucine-rich proteoglycan decorin, modifies both the direction and the extent of these changes. As the dura mater is the most important route for the brain metabolic clearance, these results are of clinical relevance and provide a much-needed link explaining the association between ophthalmic conditions and the development of neurodegenerative diseases.

Effect of Blasting Treatments on the Surface Topography and Cell Adhesion on Biodegradable FeMn‐Based Stents Processed by Laser Powder Bed Fusion

Despite all progresses made so far, in‐stent restenosis (ISR) is still a vital problem after angioplasty and stenting with permanent stents. Therefore, the effects of microblasting on laser powder bed fusion (LPBF) manufactured biodegradable Fe‐based stents with regard to surface topography and its effect on smooth muscle cell (SMC) adherence, which could be interpreted as an early hallmark for ISR, are characterized. The LPBF‐processed Fe‐30Mn‐1C‐0.025S stents are microblasted with spherical glass beads and angular corundum particles. On the microscale, the partially molten particles on the stents are significantly reduced after the surface treatments, especially after microblasting with glass beads. Angular corundum particles lead to a rougher surface on the nanoscale as demonstrated by scanning electron microscopy and atomic force microscopy. With the aim to reduce migration and proliferation of SMC, which contribute to ISR after stenting, the interactions of microblasted stent surfaces with SMC are assessed by fluorescence microscopy. Both microblasted surfaces reduce SMC adhesion and change SMC morphology compared to the as‐built state as well as to commercially available 316L stents. In conclusion, microblasting treatment shows a high potential for the postprocessing of additively manufactured, biodegradable stents due to the reduction of the surface roughness and possible beneficial effect regarding ISR.

Abstract P054: Loss Of The Smooth-muscle-cell-angiotensin Ii-sensitive Lncrna Leads To Smc Hypertrophic And Hypertensive Remodeling Due To Cell Cycle Dysregulation.

Vascular smooth muscle cells (SMC) responses to increased levels of Ang II during hypertensive remodeling included enhanced vasoconstriction and hypertrophy due to the enlargement of the SMC on the vessel wall. Hypertrophic SMC usually present defects in cell division and cell polyploidy. However, the molecular mechanisms controlling SMC hypertrophy and polyploidy vs. hyperplasia are still not fully understood. Long-non-coding-RNAs (LncRNAs) are epigenetic regulators of gene expression influencing biological processes, including cell division fidelity. We discovered a novel lncRNA decreased in dedifferentiated SMC, the SMC-Ang II-Sensitive (SAS) lncRNA, which expression is reduced in response to Ang-II in cultured SMC and the aorta of hypertensive mice, suggesting a role in mediating hypertension-induced SMC hypertrophy. Publicly available transcriptional datasets revealed that SAS is preferentially expressed in SMC-rich tissues, including the aorta and renal artery, in humans and mice. Yet, the functional relevance of SAS in SMC has never been investigated. Knockdown of SAS reduces proliferation and migration in SMC treated with Platelet-Derived Growth Factor (PDGF-BB). SAS knockdown was also associated with distinct SMC hypertrophic morphological changes, including enlargement in cell size and polynucleation in vitro. We have generated a SAS KO mouse, and aortas from these mice present a higher number of polynucleated medial SMC than their WT littermates. These data correlate with a cell cycle arrest in G1 and senescence phenotype that SAS deficient SMC present due to dysregulation in cyclins expression. Furthermore, SAS defective cells show mitochondria hyperfusion and increased oxygen consumption that correlates with the observed senescence and arrest on the G1/S checkpoint, and it is exacerbated by treatment with Ang-II. Interestingly, treatment with Losartan, an Ang-II receptor inhibitor, rescues SAS expression on Ang-II treated SMC and diminishes SMC hypertrophy. Together, these observations suggest that a decrease in SAS causes SMC hypertrophy due to defects in cell cycle completion. SAS is a potent regulator of SMC morphology and is required for proper cell division and mitochondria organization.

Regulation of extracellular matrix composition by fibroblasts during perinatal cardiac maturation

BackgroundCardiac fibroblasts are the main non-myocyte population responsible for extracellular matrix (ECM) production. During perinatal development, fibroblast expansion coincides with the transition from hyperplastic to hypertrophic myocardial growth. Therefore, we investigated the consequences of fibroblast loss at the time of cardiomyocyte maturation by depleting fibroblasts in the perinatal mouse. Methods and resultsWe evaluated the microenvironment of the perinatal heart in the absence of fibroblasts and the potential functional impact of fibroblast loss in regulation of cardiomyocyte cell cycle arrest and binucleation. Cre-mediated expression of diphtheria toxin A in PDGFRα expressing cells immediately after birth eliminated 70–80% of the cardiac fibroblasts. At postnatal day 5, hearts lacking fibroblasts appeared similar to controls with normal morphology and comparable numbers of endothelial and smooth muscle cells, despite a pronounced reduction in fibrillar collagen. Immunoblotting and proteomic analysis of control and fibroblast-deficient hearts identified differential abundance of several ECM proteins. In addition, fibroblast loss decreased tissue stiffness and resulted in increased cardiomyocyte mitotic index, DNA synthesis, and cytokinesis. Moreover, decellularized matrix from fibroblast-deficient hearts promoted cardiomyocyte DNA replication. While cardiac architecture was not overtly affected by fibroblast reduction, few pups survived past postnatal day 11, suggesting an overall requirement for PDGFRα expressing fibroblasts. ConclusionsThese studies demonstrate the key role of fibroblasts in matrix production and cardiomyocyte cross-talk during mouse perinatal heart maturation and revealed that fibroblast-derived ECM may modulate cardiomyocyte maturation in vivo. Neonatal depletion of fibroblasts demonstrated that although hearts can tolerate reduced ECM composition, fibroblast loss eventually leads to perinatal death as the approach simultaneously reduced fibroblast populations in other organs.

Effects of Pulsatility on Arterial Endothelial and Smooth Muscle Cells

Continuous flow ventricular assist device (CFVAD) support in advanced heart failure patients causes diminished pulsatility, which has been associated with adverse events including gastrointestinal bleeding, end organ failure, and arteriovenous malformation. Recently, pulsatility augmentation by pump speed modulation has been proposed as a means to minimize adverse events. Pulsatility primarily affects endothelial and smooth muscle cells in the vasculature. To study the effects of pulsatility and pulse modulation using CFVADs, we have developed a microfluidic co-culture model with human aortic endothelial (ECs) and smooth muscle cells (SMCs) that can replicate physiologic pressures, flows, shear stresses, and cyclical stretch. The effects of pulsatility and pulse frequency on ECs and SMCs were evaluated during (1) normal pulsatile flow (120/80 mmHg, 60 bpm), (2) diminished pulsatility (98/92 mmHg, 60 bpm), and (3) low cyclical frequency (115/80 mmHg, 30 bpm). Shear stresses were estimated using computational fluid dynamics (CFD) simulations. While average shear stresses (4.2 dynes/cm²) and flows (10.1 mL/min) were similar, the peak shear stresses for normal pulsatile flow (16.9 dynes/cm²) and low cyclic frequency (19.5 dynes/cm²) were higher compared to diminished pulsatility (6.45 dynes/cm²). ECs and SMCs demonstrated significantly lower cell size with diminished pulsatility compared to normal pulsatile flow. Low cyclical frequency resulted in normalization of EC cell size but not SMCs. SMCs size was higher with low frequency condition compared to diminished pulsatility but did not normalize to normal pulsatility condition. These results may suggest that pressure amplitude augmentation may have a greater effect in normalizing ECs, while both pressure amplitude and frequency may be required to normalize SMCs morphology. The co-culture model may be an ideal platform to study flow modulation strategies.

Using Polyacrylamide Hydrogels to Model Physiological Aortic Stiffness Reveals that Microtubules Are Critical Regulators of Isolated Smooth Muscle Cell Morphology and Contractility.

Vascular smooth muscle cells (VSMCs) are the predominant cell type in the medial layer of the aortic wall and normally exist in a quiescent, contractile phenotype where actomyosin-derived contractile forces maintain vascular tone. However, VSMCs are not terminally differentiated and can dedifferentiate into a proliferative, synthetic phenotype. Actomyosin force generation is essential for the function of both phenotypes. Whilst much is already known about the mechanisms of VSMC actomyosin force generation, existing assays are either low throughput and time consuming, or qualitative and inconsistent. In this study, we use polyacrylamide hydrogels, tuned to mimic the physiological stiffness of the aortic wall, in a VSMC contractility assay. Isolated VSMC area decreases following stimulation with the contractile agonists angiotensin II or carbachol. Importantly, the angiotensin II induced reduction in cell area correlated with increased traction stress generation. Inhibition of actomyosin activity using blebbistatin or Y-27632 prevented angiotensin II mediated changes in VSMC morphology, suggesting that changes in VSMC morphology and actomyosin activity are core components of the contractile response. Furthermore, we show that microtubule stability is an essential regulator of isolated VSMC contractility. Treatment with either colchicine or paclitaxel uncoupled the morphological and/or traction stress responses of angiotensin II stimulated VSMCs. Our findings support the tensegrity model of cellular mechanics and we demonstrate that microtubules act to balance actomyosin-derived traction stress generation and regulate the morphological responses of VSMCs.

Pulmonary arteries of Williams syndrome patients exhibit altered serotonin metabolism genes and degenerated medial layer architecture.

Williams-Beuren syndrome (WS) is characterized by cardiovascular abnormalities associated with a multigene deletion on 7q11.23, in particular elastin (ELN). Peripheral pulmonary artery stenosis (PPAS) frequently affects pediatric patients with WS. Molecular investigation of WS pulmonary arterial (PA) tissue is limited by tissue scarcity. We compared transcriptomes, tissue architecture, and localized changes in protein expression in PA tissue from patients with WS (n = 8) and donors (n = 5). Over 100 genes were differentially expressed at the ≥4-fold level, including genes related to the serotonin signaling pathway: >60-fold downregulation of serotonin transporter SLC6A4 and >3-fold upregulation of serotonin receptor HTR2A. Histologic examination revealed abnormal elastin distribution and smooth muscle cell morphology in WS PA, with markedly shorter, disorganized elastin fibers, and expanded proteoglycan-rich extracellular matrix between muscle layers. There were significant abnormalities in the PA expression of genes regulating serotonin signaling, metabolism, and receptors in WS. Those changes were associated with distinct changes in the arterial structure and may play a role in the stenosis-promoting effects of elevated shear stress at PA bifurcations in WS. Serotonin pathway signaling is significantly altered in the pulmonary arteries of patients with Williams syndrome and severe peripheral arterial stenosis. The present study compares the histological and biochemical characteristics of pulmonary arteries from patients with Williams syndrome to those of controls, something that has not, to our knowledge, been done previously. It demonstrates marked abnormalities in the pulmonary arteries of patients with Williams syndrome, especially significant pathologic alterations in the signaling of the serotonin pathway. The findings of this study provide direction for the development of potential therapies to treat pulmonary artery stenosis in patients with Williams syndrome.

Abstract 15560: Induced Pluripotent Stem Cell (iPSC) Derived Smooth Muscle Cells as a Model to Study Vascular Calcification

Introduction: Vascular calcification is the top cause of mortality worldwide. Vascular smooth muscle cells (SMCs) are major contributors to vascular calcification. There is an urgent need for a large scale and inexhaustible source of functional SMCs to study mechanisms of cardiovascular calcification. Here, we describe a new platform utilizing iPSCs differentiated towards SMCs that can calcify, to model ectopic calcification and identify mechanisms of SMCs’ contribution in this pathological process. Methods: We differentiated human iPSCs towards an induced-SMC (iSMC) phenotype in a 10-day protocol. We verified successful iSMC differentiation through immunofluorescent staining for SMC marker proteins αSMA and MYH11, FACS using CD140b, and proteomics analysis on whole cell lysate at days 0, 3, 5, 7, and 10. A set of 19 canonical SMC proteins were used to generate a Pearson’s correlation over the differentiation time course. After differentiation, we cultured the iSMCs in osteogenic media (OM) for 14 days. Alizarin Red S staining performed on day 14 confirmed the presence of calcification. Three independent experiments were performed for all data analyses. Results: Typical SMC morphology with positive staining for αSMA and MYH11 was detected at day 10 by immunofluorescence (Fig 1A). FACS analysis on markers for iPSCs (TRA-1-60), ECs (CD31), and SMCs (CD140b) demonstrated that 93.9% of cells were positive for CD140b after 10 days of differentiation (Fig 1B, C). Time course proteomics showed a strong Pearson’s correlation with of iSMCs with SMC marker proteins (R = 0.89) by day 10 (Figure 1D). Calcification in osteogenic media was observed by day 14 in culture using Alizarin Red S staining (Fig 1E, F), with 4.8-fold higher calcification in OM as compared to normal media (NM). Conclusions: Investigated iSMCs have promising future applications in cardiovascular research and may serve as an inexhaustible resource for studying vascular SMC calcification.

IGFBP6 regulates vascular smooth muscle cell proliferation and morphology via cyclin E-CDK2.

Despite the high prevalence of varicose veins, the underlying pathogenesis of this disease remains unclear. The present study aims to explore the role of insulin-like growth factor binding protein 6 (IGFBP6) in vascular smooth muscle cells (VSMCs). Using a protein array approach, we identified several differentially expressed proteins between varicose great saphenous veins and normal great saphenous veins. Bioinformatic analysis showed that IGFBP6 was closely related to cell proliferation. Further validation confirmed that IGFBP6 was one of the most highly expressed proteins in varicose vein tissue. Knocking down IGFBP6 in VSMCs significantly attenuated cell proliferation and induced the S phase arrest during the cell cycle. Further experiments demonstrated that IGFBP6 knockdown increased cyclin E ubiquitination, which reduced expression of cyclin E and phosphorylation of CDK2. Furthermore, IGFBP6 knockdown arrested centrosome replication, which subsequently influenced VSMC morphology. Ultimately, IGFBP6 was validated to be involved in VSMC proliferation in varicose vein tissues. The present study reveals that IGFBP6 is closely correlated with VSMC biological function and provides unprecedented insights into the underlying pathogenesis of varicose veins.

Traditional Chinese medicine insights of newly-diagnosed and young hypertension and clinical practice of Tianma Gouteng Decoction for hypertension treatment

Hypertension is a global major public health problem. Early intervention and timely blocking are necessary for newly-diagnosed hypertensive and young hypertensive patients. Tianma Gouteng Decoction recorded in Zabing Zhengzhi Xinyi written by HU Guangci has the major functions in treating excess syndrome and wind-Yang disturbance syndrome with effects in calming liver-wind, clearing heat and activating blood, and nourishing liver and kidney. As we known, Tianma Gouteng Decoction is a classical formula for treating hypertension. However, due to the limitation of historical conditions, some problems impede Tianma Gouteng Decoction from being handed down successfully, such as uncertain diagnosis, uncertain antihypertensive efficacy and treatment-course. In this case, we ask whether Tianma Gouteng Decoction can be used as antihypertensive therapy and the exactly decrease of systolic and diastolic blood pressure, and wonder the half-life of Tianma Gouteng Decoction and the relationship with Western medicine. Therefore, defined prescription and syndrome, efficacy, treatment-course are important in clinical practice. According to the findings, ① previously, we thought that hypertension was a liver-Yang hyperactivity and liver-wind stirring syndrome, and the liver-based therapy theory was effective. However, based on our study, long-term treatment with Western medicine could block disease progression, control the increased blood pressure and change the pathogenesis and syndrome of traditional Chinese medicine. Therefore, the frequency of liver-Yang hyperactivity syndrome "fire syndrome" decreased sharply, and the main syndrome was fluid-retention syndrome and deficiency syndrome. The etiology and pathogenesis of hypertension was prone to deficiency syndrome accompanied with excess syndrome. Therefore, it was necessary to intervene hypertension in "liver-wind" and "liver-Yang" excess syndrome stage, and the early intervention and treatment are important. ② The newly-diagnosed hypertensive and young hypertensive patients usually had no Western medicine intervention. Thus, the etiology and pathogenesis of hypertension were free from Western medicine, which maintained the initial stage of hypertension in line with the liver-Yang hyperactivity syndrome. ③ The pathological mechanisms of newly-diagnosed and young hypertension were mainly increase in sympathetic activity, active renin-angiotensin-aldosterone system, change in morphology of vascular smooth muscle cells and endothelial cell dysfunction. ④ According to the syndrome of Tianma Gouteng Decoction, in modern medicine, Tianma Gouteng Decoction was widely used to treat the early-stage hypertension and the early-intervention of hypertension without Western medicine, particularly newly-diagnosed, young hypertension, Ⅲ hypertension, hypertensive crisis and hypertensive emergency. Tianma Gouteng Decoction can treat such symptoms as headache, dizziness, fullness of head, slurring of speech; facial flushing, conjunctival congestion and blurred vision; dysphoria, anxiety, palpitation, insomnia, mental disorder and hot flushes; dry-mouth with bitter taste and sweeting; myasthenia of limbs and lassitude in loin and legs; brown urine with burning sensation during urination; a solid or liquid stool consistency; red tongue, slippery and rapid pulse, wiry and rapid pulse over to Cunkou. ⑤ After treatment with Tianma Gouteng Decoction for three to six months, the blood pressure was decreased, and the symptoms were improved with reduction or even discontinuance of Western medicine. ⑥ According to recent studies, Caul is Polygoni Multiflori of Tianma Gouteng Decoction induced certain hepatotoxicity and increased the risk of long-term treatment. However, with the regular monitoring of biomedical index, long-term treatment with Caulis Polygoni Multiflori(3-10 g·d~(-1) for 3 to 6 months) didn't show hepatic dysfunction. ⑦ The pharmacological activity of Tianma Gouteng Decoction coincided with the pathology and disease mechanism of newly-diagnosed and young hypertension. Therefore, Tianma Gouteng Decoction was the specific recipe for newly-diagnosed and young hypertension. Therefore, further studies of efficacy, safety and molecular mechanism of Tianma Gouteng Decoction may make a medical breakthrough for hypertension treatment.

Detrusor bioengineering using a cell-enriched compressed collagen hydrogel.

The gold standard for bladder regeneration in end-stage bladder disease is the use of intestinal tissue, which is however associated with significant long-term complications. Our study aims to bioengineer functional detrusor muscle combining bladder smooth muscle cells (SMC) and SMC-like adipose-derived stem cells (pADSC) in compressed collagen (CC) hydrogels and to investigate biocompatibility and tissue regeneration of such detrusor-equivalents in a rat detrusorectomy model. Compressed collagen hydrogels seeded with 1 × 106 or 4 × 106 SMC alone or in combination with pADSC in a 1:1 ratio were investigated. Morphology, phenotype, and viability as well as proteomic secretome analysis were assessed in the 1:1 co-cultures and the respective monocultures. The hydrogels were implanted into rat bladders after partial detrusorectomy. Bladders were harvested 8 weeks after transplantation, and assessed for tissue morphology, detrusor regeneration, neo-vascularization and -innervation. Co-cultured cells exhibited native SMC morphology, high viability and proliferated to form microtissues in vitro. The pro-angiogenic factors angiogenin, vascular endothelial growth factor (VEGF)-A and -D were increased in the secretome of the pADSC samples. After 8 weeks of in vivo, the regenerated bladder wall showed a multilayered structure containing all bladder wall components. The overall performance of the bladder wall regeneration of CC seeded with 4 × 106 cells was significantly better than with 1 × 106 cells and the combination SMC:pADCS performed slightly better than SMC alone. Compressed collagen possesses an adequate regenerative potential to promote regeneration of bladder wall tissue in vivo. Seeded with a combination of pADSC and SMC this may well be the first step towards a functional bladder reconstruction especially in patients suffering of end-stage bladder diseases.

MiR-145 alleviates Hcy-induced VSMC proliferation, migration, and phenotypic switch through repression of the PI3K/Akt/mTOR pathway.

The proliferation, migration, and cellular morphology of vascular smooth muscle cells (VSMCs) play important roles in the pathogenesis of atherosclerosis (AS). Homocysteine (Hcy) is a sulfur-containing amino acid, which is an intermediate product of methionine metabolism. Hcy can induce proliferation, migration, and phenotypic switch of VSMCs, but details of these mechanisms are still unclear. The phosphatidylinositol 3-kinase (PI3K/Akt/mTOR) signaling pathway is involved in a host of cellular functions. In this study, we sought to determine if this multifunctional pathway played a role in Hcy-induced proliferation, migration, and phenotypic transformation of VSMCs, which has not been previously reported. miR-145 has been previously reported to suppress the effects of Hcy in VSMCs. In our study, using qRT-PCR, we found that Hcy itself reduced the expression of miR-145 in VSMCs, while overexpression of miR-145 reduced the proliferation, migration, and phenotypic transformation of VSMCs caused by Hcy. Using Western blot analysis, we found that VSMCs exposed to Hcy exhibited significant increases in the levels of PI3K, Akt, and mTOR proteins. Additionally, overexpression of miR-145 dramatically decreased PI3K, Akt, and mTOR expression. Using qRT-PCR we found that miR-145 expression increased after blocking PI3K using an inhibitor. Inhibition of the PI3K signaling pathway also prevented Hcy-induced VSMC proliferation, migration, and phenotypic switch. Taken together, our results suggest that miR-145 could inhibit VSMC proliferation, migration, and phenotype switching by preventing activation of the PI3K/Akt/mTOR signaling pathway.

A tripartite cooperative mechanism confers resistance of the protein kinase A catalytic subunit to dephosphorylation

Phosphorylation of specific residues in the activation loops of AGC kinase group (protein kinase A, G, and C families) is required for activity of most of these kinases, including the catalytic subunit of PKA (PKAc). Although many phosphorylated AGC kinases are sensitive to phosphatase-mediated dephosphorylation, the PKAc activation loop uniquely resists dephosphorylation, rendering it "constitutively" phosphorylated in cells. Previous biophysical experiments and structural modeling have suggested that the N-terminal myristoylation signal and the C-terminal FXXF motif in PKAc regulate its thermal stability and catalysis. Here, using site-directed mutagenesis, molecular modeling, and in cell-free and cell-based systems, we demonstrate that substitutions of either the PKAc myristoylation signal or the FXXF motif only modestly reduce phosphorylation and fail to affect PKAc function in cells. However, we observed that these two sites cooperate with an N-terminal FXXW motif to cooperatively establish phosphatase resistance of PKAc while not affecting kinase-dependent phosphorylation of the activation loop. We noted that this tripartite cooperative mechanism of phosphatase resistance is functionally relevant, as demonstrated by changes in morphology, adhesion, and migration of human airway smooth muscle cells transfected with PKAc variants containing amino acid substitutions in these three sites. These findings establish that three allosteric sites located at the PKAc N and C termini coordinately regulate the phosphatase sensitivity of this enzyme. This cooperative mechanism of phosphatase resistance of AGC kinase opens new perspectives toward therapeutic manipulation of kinase signaling in disease.

A Novel in situ Approach to Studying Detrusor Smooth Muscle Cells in Mice

The aim of our study was to develop a novel approach to investigating mouse detrusor smooth muscle cell (SMC) physiological activity, utilizing an acute tissue dissection technique and confocal calcium imaging. The bladder of a sacrificed adult female NMRI mouse was dissected. We used light and transmission electron microscopy to assess morphology of SMCs within the tissue. Calcium imaging in individual SMCs was performed using confocal microscopy during stimulation with increasing concentrations of carbamylcholine (CCh). SMCs were identified according to their morphology and calcium activity. We determined several parameters describing the SMC responses: delays to response, recruitment, relative activity, and contraction of the tissue. CCh stimulation revealed three different SMC phenotypes: spontaneously active SMCs with and without CCh-enhanced activity and SMCs with CCh-induced activity only. SMCs were recruited into an active state in response to CCh-stimulation within a narrow range (1–25 µM); causing activation of virtually all SMCs. Maximum calcium activity of SMCs was at about 25 µM, which coincided with a visible tissue contraction. Finally, we observed shorter time lags before response onsets with higher CCh concentrations. In conclusion, our novel in situ approach proved to be a robust and reproducible method to study detrusor SMC morphology and physiology.

Effects of cyclic stretch on nuclear morphology of vascular smooth muscle cells in 3D substrates

Effects of cyclic stretch on nuclear morphology of vascular smooth muscle cells in 3D substrates

Histological study on the safety of the controllable ileostomy with pipe

To investigate the safety of the controllable ileostomy with pipe in view of histology. Twenty-eight Beagle dogs undergoing controllable ileostomy with pipe were studied. The special fistula tube with balloon was placed into the hole locating at the cecal root opposing the mesenteric side, and fixed by double knot compression method. The fistula tube was removed 14 days after surgery, then the safety of the procedure was preliminarily evaluated by gastrointestinal radiography and anatomical observation. The small intestine tissue at the compression suture was used as the experimental segment, and the small intestine tissue at the proximal non-compression suture was used as the control segment. The histological staining and the immunohistochemical staining of S-100 protein, c-kit protein and α-smooth muscle actin(α-SMA) protein between two segment were compared, while quantitative comparison of myenteric plexus, intestinal Cajal cell(ICC) and smooth muscle cells in intestinal wall was carried out. After removal of fistula tube at 14 days postoperative, the dogs were normal in feeding and defecation. The digestive tract radiography showed that the intestine was patent without obvious stenosis and obstruction. The dogs were dissected 21 days after operation. The abdominal sinus ostium was well healed and the internal sinus was well formed. Under gross inspection, blood supply, morphology and motor function of experimental intestine segment were similar from the proximal and distal segments of control intestine. S-100 immunohistochemical staining showed that the morphology and distribution of S-100 protein positive cells and "blank area" cells in the experimental and control segments were consistent. Myenteric plexus counting showed that the experimental segment was 3.62±1.82/field and the control segment was 3.27±1.62/field, whose difference was not statistically significant(t=1.30, P=0.20). Immunohistochemical staining of c-kit showed that the distribution of c-kit positive cells in both segments was consistent. Counting of the number of ICCs in myenteric plexus revealed that experimental segment was 2.96±2.57/plexus, and control segment was 2.49±1.80/plexus without significant difference(t=1.81, P=0.07). Immunohistochemical staining of α-SMA showed that the morphology and distribution of smooth muscle cells in whole intestinal wall(muscle layer, longitudinal muscle, ring muscle) in experimental and control segments were consistent. The average absorbance(A) value of α-SMA staining in ring muscle layer was detected and quantified. The experimental segment was 0.15±0.03 and control segment was 0.14±0.04 without significant difference(t=1.16, P=0.25). The technique of controllable ileostomy with pipe is safe in view of histology, which may replace the traditional protective ileostomy.

The effects of substrate stiffness on the morphology, intercellular tension, and differentiation of vascular smooth muscle cells (Analysis of the surface microstructure and tension of cells using atomic force microscopy)

The effects of substrate stiffness on the morphology, intercellular tension, and differentiation of vascular smooth muscle cells (Analysis of the surface microstructure and tension of cells using atomic force microscopy)

Platelet extracellular vesicles induce a pro-inflammatory smooth muscle cell phenotype

ABSTRACTExtracellular vesicles (EVs) are mediators of cell communication during health and disease, and abundantly released by platelets upon activation or during ageing. Platelet EVs exert modulatory effects on immune and vascular cells. Platelet EVs may modulate the function of vascular smooth muscle cells (SMC). Platelet EVs were isolated from platelet-rich plasma and incubated with SMC in order to assess binding, proliferation, migration and pro-inflammatory phenotype of the cells. Platelet EVs firmly bound to resting SMC through the platelet integrin αIIbβ3, while binding also occurred in a CX3CL1–CX3CR1-dependent manner after cytokine stimulation. Platelet EVs increased SMC migration comparable to platelet derived growth factor or platelet factor 4 and induced SMC proliferation, which relied on CD40- and P-selectin interactions. Flow-resistant monocyte adhesion to platelet EV-treated SMC was increased compared with resting SMC. Again, this adhesion depended on integrin αIIbβ3 and P-selectin, and to a lesser extent on CD40 and CX3CR1. Treatment of SMC with platelet EVs induced interleukin 6 secretion. Finally, platelet EVs induced a synthetic SMC morphology and decreased calponin expression. Collectively, these data indicate that platelet EVs exert a strong immunomodulatory activity on SMC. In particular, platelet EVs induce a switch towards a pro-inflammatory phenotype, stimulating vascular remodelling.

Higenamine inhibits apoptosis and maintains survival of gastric smooth muscle cells in diabetic gastroparesis rat model via activating the β2-AR/PI3K/AKT pathway

Diabetic gastroparesis (DGP) is a common complication of diabetes mellitus (DM). The numerous clinical symptoms of DGP and the great cost on the treatment of DGP seriously lowered the patients’ life quality. However, the pathogenic mechanism of DGP is still elusive till now. In this study, we aimed to explore the effect of higenamine on the proliferation and apoptosis of gastric smooth muscle cells (SMCs) in DGP rat model. The DGP rat model was built by intraperitoneal injection of Streptozotocin (STZ) into male Sprague-Dawley (SD) rats. Compared with the healthy control group, the level of DGP indicator c-kit was strongly suppressed and the level of Gsα was largely elevated in the STZ-induced model group. By contrast, the addition of higenamine obviously counteracted the effect of STZ on the expression of c-kit and Gsα. Besides that, higenamine improved the decreased emptying ability of the stomach. In addition, the number of gastric SMCs was strongly decreased and cell morphology became irregular in STZ-induced model group. The treatment of higenamine weakened the harm of STZ on the number and morphology of gastric SMCs. Beyond that, higenamine promoted gastric SMCs proliferation and inhibited gastric SMCs apoptosis in DGP model. Further research revealed that higenamine regulated cell proliferation and apoptosis via activating the β2-AR/PI3K/AKT pathway. Taken together, our research revealed that higenamine maintained the survival of gastric SMCs in DGP rat model via the β2-AR/PI3K/AKT pathway, providing a new sight for the treatment of DGP.