Heterogeneity Of Smooth Muscle Cells Research Articles

Deciphering smooth muscle cell heterogeneity in atherosclerotic plaques and constructing model: a multi-omics approach with focus on KLF15/IGFBP4 axis

BackgroundRuptured atherosclerotic plaques often precipitate severe ischemic events, such as stroke and myocardial infarction. Unraveling the intricate molecular mechanisms governing vascular smooth muscle cell (VSMC) behavior in plaque stabilization remains a formidable challenge.MethodsIn this study, we leveraged single-cell and transcriptomic datasets from atherosclerotic plaques retrieved from the gene expression omnibus (GEO) database. Employing a combination of single-cell population differential analysis, weighted gene co-expression network analysis (WGCNA), and transcriptome differential analysis techniques, we identified specific genes steering the transformation of VSMCs in atherosclerotic plaques. Diagnostic models were developed and validated through gene intersection, utilizing the least absolute shrinkage and selection operator (LASSO) and random forest (RF) methods. Nomograms for plaque assessment were constructed. Tissue localization and expression validation were performed on specimens from animal models, utilizing immunofluorescence co-localization, western blot, and reverse-transcription quantitative-polymerase chain reaction (RT-qPCR). Various online databases were harnessed to predict transcription factors (TFs) and their interacting compounds, with determination of the cell-specific localization of TF expression using single-cell data.ResultsFollowing rigorous quality control procedures, we obtained a total of 40,953 cells, with 6,261 representing VSMCs. The VSMC population was subsequently clustered into 5 distinct subpopulations. Analyzing inter-subpopulation cellular communication, we focused on the SMC2 and SMC5 subpopulations. Single-cell subpopulation and WGCNA analyses revealed significant module enrichments, notably in collagen-containing extracellular matrix and cell-substrate junctions. Insulin-like growth factor binding protein 4 (IGFBP4), apolipoprotein E (APOE), and cathepsin C (CTSC) were identified as potential diagnostic markers for early and advanced plaques. Notably, gene expression pattern analysis suggested that IGFBP4 might serve as a protective gene, a hypothesis validated through tissue localization and expression analysis. Finally, we predicted TFs capable of binding to IGFBP4, with Krüppel-like family 15 (KLF15) emerging as a prominent candidate showing relative specificity within smooth muscle cells. Predictions about compounds associated with affecting KLF15 expression were also made.ConclusionOur study established a plaque diagnostic and assessment model and analyzed the molecular interaction mechanisms of smooth muscle cells within plaques. Further analysis revealed that the transcription factor KLF15 may regulate the biological behaviors of smooth muscle cells through the KLF15/IGFBP4 axis, thereby influencing the stability of advanced plaques via modulation of the PI3K-AKT signaling pathway. This could potentially serve as a target for plaque stability assessment and therapy, thus driving advancements in the management and treatment of atherosclerotic plaques.

Embryonic Heterogeneity of Smooth Muscle Cells in the Complex Mechanisms of Thoracic Aortic Aneurysms.

Smooth muscle cells (SMCs) are the major cell type of the aortic wall and play a pivotal role in the pathophysiology of thoracic aortic aneurysms (TAAs). TAAs occur in a region-specific manner with the proximal region being a common location. In this region, SMCs are derived embryonically from either the cardiac neural crest or the second heart field. These cells of distinct origins reside in specific locations and exhibit different biological behaviors in the complex mechanism of TAAs. The purpose of this review is to enhance understanding of the embryonic heterogeneity of SMCs in the proximal thoracic aorta and their functions in TAAs.

Vascular Smooth Muscle Cell Subpopulations and Neointimal Formation in Mouse Models of Elastin Insufficiency.

Using a mouse model of Eln (elastin) insufficiency that spontaneously develops neointima in the ascending aorta, we sought to understand the origin and phenotypic heterogeneity of smooth muscle cells (SMCs) contributing to intimal hyperplasia. We were also interested in exploring how vascular cells adapt to the absence of Eln. Approach and Results: We used single-cell sequencing together with lineage-specific cell labeling to identify neointimal cell populations in a noninjury, genetic model of neointimal formation. Inactivating Eln production in vascular SMCs results in rapid intimal hyperplasia around breaks in the ascending aorta's internal elastic lamina. Using lineage-specific Cre drivers to both lineage mark and inactivate Eln expression in the secondary heart field and neural crest aortic SMCs, we found that cells with a secondary heart field lineage are significant contributors to neointima formation. We also identified a small population of secondary heart field-derived SMCs underneath and adjacent to the internal elastic lamina. Within the neointima of SMC-Eln knockout mice, 2 unique SMC populations were identified that are transcriptionally different from other SMCs. While these cells had a distinct gene signature, they expressed several genes identified in other studies of neointimal lesions, suggesting that some mechanisms underlying neointima formation in Eln insufficiency are shared with adult vessel injury models. These results highlight the unique developmental origin and transcriptional signature of cells contributing to neointima in the ascending aorta. Our findings also show that the absence of Eln, or changes in elastic fiber integrity, influences the SMC biological niche in ways that lead to altered cell phenotypes.

Features of Remodeling of Prostate Stroma in Chronic Abacterial Prostatitis.

We performed a complex morphological study of biopsy samples of prostate gland from 30 men (mean age 46±11 years) with chronic abacterial prostatitis who were exposed unfavorable anthropogenic factors. The ultrastructural heterogeneity of smooth muscle cells was shown, among which cells with destructive changes in organelles predominated, solitary cells with signs of secretory function were revealed. Most endothelial cells in microvessels were characterized by low pinocytotic activity, destruction of organelles; focal hyperplastic ultrastructural changes were noted in some endotheliocytes. Along with fibroblasts, the stroma contained single mast cells; no inflammatory infiltration was revealed. The results of the study attested to complex mechanisms of remodeling of the prostate gland stroma in chronic abacterial prostatitis involving microvessels, smooth muscle cells, and probably mast cells, which determines the development of degenerative, destructive, and fibrotic processes.

Implication of molecular vascular smooth muscle cell heterogeneity among arterial beds in arterial calcification.

Vascular calcification is a strong and independent predictive factor for cardiovascular complications and mortality. Our previous work identified important discrepancies in plaque composition and calcification types between carotid and femoral arteries. The objective of this study is to further characterize and understand the heterogeneity in vascular calcification among vascular beds, and to identify molecular mechanisms underlying this process. We established ECLAGEN biocollection that encompasses human atherosclerotic lesions and healthy arteries from different locations (abdominal, thoracic aorta, carotid, femoral, and infrapopliteal arteries) for histological, cell isolation, and transcriptomic analysis. Our results show that lesion composition differs between these locations. Femoral arteries are the most calcified arteries overall. They develop denser calcifications (sheet-like, nodule), and are highly susceptible to osteoid metaplasia. These discrepancies may derive from intrinsic differences between SMCs originating from these locations, as microarray analysis showed specific transcriptomic profiles between primary SMCs isolated from each arterial bed. These molecular differences translated into functional disparities. SMC from femoral arteries showed the highest propensity to mineralize due to an increase in basal TGFβ signaling. Our results suggest that biological heterogeneity of resident vascular cells between arterial beds, reflected by our transcriptomic analysis, is critical in understanding plaque biology and calcification, and may have strong implications in vascular therapeutic approaches.

Mouse-to-mouse variation in maturation heterogeneity of smooth muscle cells.

Smooth muscle cell (SMC) heterogeneity plays an important role in vascular remodeling, a life-threatening hallmark of many vascular diseases. However, the characterization of SMCs at the single-cell level is stymied by drawbacks of contemporary single-cell protein measurements, including antibody probe cross-reactivity, chemical fixation artifacts, limited isoform-specific probes, low multiplexing and difficulty sampling cells with irregular morphologies. To scrutinize healthy vessels for subpopulations of SMCs with proliferative-like phenotypes, we developed a high-specificity, multiplexed single-cell immunoblotting cytometry tool for unfixed, uncultured primary cells. We applied our assay to demonstrate maturation stage profiling of aortic SMCs freshly isolated from individual mice. After ensuring unbiased sampling of SMCs (80-120 μm in length), we performed single-SMC electrophoretic protein separations, which resolve protein signal from off-target antibody binding, and immunoblotted for differentiation markers α-SMA, CNN-1 and SMMHC (targets ranging from 34 kDa to 227 kDa). We identified a subpopulation of immature-like SMCs, supporting the recently-established mechanism that only a subset of SMCs is responsible for vascular remodeling. Furthermore, the low sample requirements of our assay enable single-mouse resolution studies, which minimizes animal sacrifice and experimental costs while reporting animal-to-animal phenotypic variation, essential for achieving reproducibility and surmounting the drawbacks of pooling primary cells from different animals.

207 Vascular smooth muscle cell heterogeneity and plasticity

IntroductionVascular smooth muscle cells (VSMCs) show inherent plasticity, enabling their phenotypic switch into a synthetic state for vascular repair and remodelling. Under inflammatory conditions, this contributes to atherosclerotic plaque development,...

Effects of Fluid Shear Stress on a Distinct Population of Vascular Smooth Muscle Cells

Vascular smooth muscle cells (SMCs) are a major cell type involved in vascular remodeling. The various developmental origins of SMCs such as neural crest and mesoderm result in heterogeneity of SMCs, which plays an important role in the development of vascular remodeling and diseases. Upon vascular injury, SMCs are exposed to blood flow and subjected to fluid shear stress. Previous studies have shown that fluid shear stress inhibits SMC proliferation. However, the effect of shear stress on the subpopulation of SMCs from specific developmental origin and vascular bed is not well understood. Here we investigated how shear stress regulates human aortic SMCs positive for neural crest markers. DNA microarray analysis showed that shear stress modulates the expression of genes involved in cell proliferation, matrix synthesis, cell signaling, transcription and cytoskeleton organization. Further studies demonstrated that shear stress induced SMC proliferation and cyclin D1, downregulated cell cycle inhibitor p21, and activated Akt pathway. Inhibition of PI-3 kinase blocked these shear stress-induced changes. These results suggest that SMCs with neural crest characteristics may respond to shear stress in a different manner. This finding has significant implications in the remodeling and diseases of blood vessels.

11 Generation of developmental origin-specific vascular smooth muscle cells from human embryonic stem cells

Heterogeneity is a hallmark of vascular smooth muscle cell (SMC) development, physiology and pathology. This could be attributed to the diverse embryological origins of vascular SMCs. In order to uncover...

Smooth Muscle Progenitor Cells: Friend or Foe in Vascular Disease?

The origin of vascular smooth muscle cells that accumulate in the neointima in vascular diseases such as transplant arteriosclerosis, atherosclerosis and restenosis remains subject to much debate. Smooth muscle cells are a highly heterogeneous cell population with different characteristics and markers, and distinct phenotypes in physiological and pathological conditions. Several studies have reported a role for bone marrow-derived progenitor cells in vascular maintenance and repair. Moreover, bone marrow-derived smooth muscle progenitor cells have been detected in human atherosclerotic tissue as well as in in vivo mouse models of vascular disease. However, it is not clear whether smooth muscle progenitor cells can be regarded as a ‘friend’ or ‘foe’ in neointima formation. In this review we will discuss the heterogeneity of smooth muscle cells, the role of smooth muscle progenitor cells in vascular disease, potential mechanisms that could regulate smooth muscle progenitor cell contribution and the implications this may have on designing novel therapeutic tools to prevent development and progression of vascular disease.

20-HETE inhibits the proliferation of vascular smooth muscle cells via transforming growth factor-β

20-Hydroxyeicosatetraenoic acid (20-HETE), a cytochrome P450 arachidonic acid metabolite, has been shown to modulate the growth of vascular smooth muscle cells (VSMCs). We asked whether 20-HETE modulates the proliferation of R22D cells, a clonal VSMC from neonatal rats, by releasing transforming growth factor-beta (TGF-beta). Incubation of R22D cells with 20-HETE for 24 h attenuated [(3)H]thymidine incorporation in a concentration-dependent manner without causing the release of lactate dehydrogenase. 20-HETE also inhibited platelet-derived growth factor (PDGF)-induced [(3)H]thymidine incorporation in R22D cells and human VSMCs. At 5 muM, 20-HETE reduced [(3)H]thymidine incorporation by 34 +/- 6%; anti-TGF-beta neutralizing antibody, but not nonspecific IgG, completely reversed the attenuated [(3)H]thymidine incorporation induced by 20-HETE. In addition, 20-HETE attenuated fetal bovine serum- and PDGF-induced expression of cyclin D1, a downstream effector of TGF-beta(1), which was reversed by anti-TGF-beta antibody. Further studies demonstrated that 20-HETE may increase TGF-beta release to a level high enough to inhibit [(3)H]thymidine incorporation without altering the steady-state mRNA level of TGF-beta. Nevertheless, pretreatment of indomethacin (a cyclooxygenase inhibitor) or paxilline (a potassium channel inhibitor) did not affect the inhibitory effect on DNA synthesis induced by 20-HETE. These results demonstrate for the first time a growth-inhibitory effect induced by 20-HETE, which may be mediated by TGF-beta.

Smooth muscle cell signal transduction: Implications of vascular biology for vascular surgeons

Vascular smooth muscle cells exhibit varied responses after vessel injury and surgical interventions, including phenotypic switching, migration, proliferation, protein synthesis, and apoptosis. Although the source of the smooth muscle cells that accumulate in the vascular wall is controversial, possibly reflecting migration from the adventitia, from the circulating blood, or in situ differentiation, the intracellular signal transduction pathways that control these processes are being defined. Some of these pathways include the Ras-mitogen-activated protein kinase, phosphatidylinositol 3-kinase-Akt, Rho, death receptor-caspase, and nitric oxide pathways. Signal transduction pathways provide amplification, redundancy, and control points within the cell and culminate in biologic responses. We review some of the signaling pathways activated within smooth muscle cells that contribute to smooth muscle cell heterogeneity and development of pathology such as restenosis and neointimal hyperplasia.

Continuous wavelet transform of laser-Doppler signals from facial microcirculation reveals vasomotion asymmetry

Facial laser Doppler flux (FLDF) consists of a mean value and complex oscillatory components called vasomotion. Vasomotion can be expressed as spectral amplitudes (SA) after the application of continuous wavelet transform. Vasomotion is influenced by cardiac activity (frequency interval I), respiration (interval II), intrinsic myogenic activity (interval III), neural mechanisms (interval IV) and endothelial mechanisms (intervals V and VI). Asymmetry of FLDF mean value was previously documented and hereby we present homogeneity assessment for FLDF vasomotion. Mean FLDF ( p < 0.001), total SA ( p < 0.001) and SA within frequency intervals I–VI were significantly higher in both cheeks compared to forehead. Total SA ( p = 0.009) and SA within frequency intervals I ( p = 0.041), II ( p = 0.005), III ( p = 0.009), IV ( p = 0.001) and V ( p = 0.036) were significantly higher in right than in left forehead. Human face is a heterogeneous microvascular region. Angiographic characteristics of deep horizontal sub-dermal plexus, endothelial and vascular smooth muscle cell heterogeneity, and plasticity of the microvasculature, autonomic asymmetry and facial neuropsychological asymmetry are possible causes of microvascular asymmetry. The origin and significance of microvascular imbalance need to be elucidated further.

Vascular-wall remodeling of 3 human bypass vessels: Organ culture and smooth muscle cell properties

Late graft occlusions after coronary artery bypass grafting have been ascribed to neointimal hyperplasia. Given the pivotal role of smooth muscle cells in the pathogenesis of neointimal hyperplasia and the phenotypic heterogeneity of smooth muscle cells across vessels, we hypothesized that differences in long-term graft patency are at least partly related to differences in smooth muscle cell properties. The aim of the present study was to compare the vascular-wall remodeling of human internal thoracic artery, radial artery, and saphenous vein bypass conduits. We evaluated the intimal thickening of the human graft segments in organ cultures (histopathology, morphometric, and immunofluorescence analyses) and assessed the properties of cultured smooth muscle cells isolated from these vessels in terms of cell proliferation (tritiated thymidine incorporation), migration (modified Boyden chamber), and collagen synthesis (tritiated proline incorporation). The total vessel-wall growth index and the intimal growth index were significantly higher for saphenous vein rings than for radial artery and internal thoracic artery rings. Immunofluorescence analyses showed predominant involvement of smooth muscle cells in neointimal growth induced by organ culture of saphenous vein rings. Cell proliferation was significantly higher in saphenous vein smooth muscle cells than in radial artery smooth muscle cells and significantly higher in radial artery smooth muscle cells than in internal thoracic artery smooth muscle cells. Migration of smooth muscle cells from saphenous vein grafts was significantly greater than from internal thoracic artery or radial artery grafts. Collagen synthesis was similar in smooth muscle cells from internal thoracic artery, radial artery, and saphenous vein grafts. Ex vivo vascular-wall remodeling and smooth muscle cell intrinsic growth and migratory properties are dissimilar between arterial and venous grafts and might shed light on reported angiographic patency rates of these grafts.

HIGHLIGHTED TOPIC: Pulmonary Circulation and Hypoxia

In 1946, Ulf von Euler and Goran Liljestrand published revolutionary findings on the impact of hypoxia as a stimulant for vasoconstriction in the pulmonary circulation. More than 50 years later, exploring the mechanistic basis for this relationship is still a vital area of research. Among the many

Heterogeneity of smooth muscle cell populations cultured from pig coronary artery.

Heterogeneous smooth muscle cell (SMC) populations have been described in the arteries of several species. We have investigated whether SMC heterogeneity is present in the porcine coronary artery, which is widely used as a model of restenosis. By using 2 isolation methods, distinct medial populations were identified: spindle-shaped SMCs (S-SMCs) after enzymatic digestion, with a "hill-and-valley" growth pattern, and rhomboid SMCs (R-SMCs) after explantation, which grow as a monolayer. Moreover, the intimal thickening that was induced after stent implantation yielded a large proportion of R-SMCs. R-SMCs exhibited high proliferative and migratory activities and high urokinase activity and were poorly differentiated compared with S-SMCs. Heparin and transforming growth factor-beta2 inhibited proliferation and increased differentiation in both populations, whereas fibroblast growth factor-2 and platelet-derived growth factor-BB had the opposite effect. In addition, S-SMCs treated with fibroblast growth factor-2 or platelet-derived growth factor-BB or placed in coculture with coronary artery endothelial cells acquired a rhomboid phenotype. This change was reversible and was also observed with S-SMC clones, suggesting that it depends on phenotypic modulation rather than on selection. Our results show that 2 distinct SMC subpopulations can be recovered from the pig coronary artery media. The study of these subpopulations will be useful for understanding the mechanisms of restenosis.

Heterogeneity of smooth muscle cells in advanced human atherosclerotic plaques: intimal smooth muscle cells expressing a fibroblast surface protein are highly activated by platelet-released products.

In vascular disease, smooth muscle cells (SMC) undergo phenotypic modulation and may acquire properties resembling those of fibroblasts in tissue wound healing. We aimed to show the differential expression of a fibroblast surface protein (FSP) by SMC in atherosclerotic lesions. In early human coronary atherosclerotic lesions the expression of FSP in the intima was absent. In contrast, 29 of 29 middle/advanced lesions contained intimal SMC expressing high levels of FSP. Fibroblast surface protein positive SMC were negative for desmin but expressed variable levels of alpha-SM actin, SM caldesmon, SM myosin heavy chain and vimentin. Explants from advanced atherosclerotic lesions yielded two main SMC subpopulations. SMC over-expressing FSP exhibited higher in vitro mitogenic response (premitotic DNA synthesis) to sera (2- to 8-fold) and platelet-released products (8- to 26-fold), especially from thrombin-activated platelets, than FSP-negative SMC. Our results suggest that the expression of FSP in SMC could indicate an activated phenotype, and the presence of highly positive FSP cells in the atherosclerotic lesions might be indicative of an increased SMC responsiveness to processes that locally generate thrombin and activate platelets.

Vascular smooth muscle and arterial calcification.

Smooth muscle cultures can calcify under certain circumstances. As a model system these cultures therefore provide information on why calcification occurs in atherosclerotic plaques. Whether all smooth muscle cells (under certain conditions), or only specific populations, can produce this mineralization has not been resolved. Demer's group has cloned calcifying vascular cells from subcultured bovine aorta and studied them in detail. They have speculated on whether the cells are smooth muscle which have altered in phenotype, or whether they are derived from a stem cell population within the artery wall. The article argues that while the normal process of smooth muscle phenotypic modulation seen in arterial repair could account for the observations, this view may be two simplistic considering the complex nature of the artery wall. Certainly there is evidence for heterogeneity of smooth muscle cells in the artery wall and recent evidence suggests that stem cells can circulate in the blood and repopulate tissues. Further studies are required to resolve the important question as to the origin of cells which produce mineralization in atheroma.

Smooth muscle cell phenotypes in atherosclerotic lesions.

Recently, there has been a dramatic change in the way we think about the role of vascular smooth muscle cells in atherosclerosis, and it is now generally accepted that a dearth of vascular smooth muscle cells in an atherosclerotic plaque is a detrimental feature of the disease. Indeed, it is now recognized that the phenotypes of vascular smooth muscle cells within a plaque dictate its features, progression and stability. Therefore an understanding of the processes that generate and regulate vascular smooth muscle cell heterogeneity are of critical importance for future therapeutic advancement in the treatment of atherosclerosis.

Smooth Muscle Cell Heterogeneity: Role of Specific Smooth Muscle Cell Subpopulations in Pulmonary Vascular Disease

The severity of chronic forms of pulmonary hypertension is determined, at least in part, by the extent of structural changes in the pulmonary arterial wall. Depending on the stimulus causing pulmonary hypertension, and on the location within the lung of the vessel examined, variable degrees of intimal, medial, and/or adventitial thickening have been observed. Intimal and medial changes are due, in large part, to smooth muscle cell (SMC) proliferation, hypertrophy, and matrix deposition. Blood-borne and locally produced mitogens, hypoxia, and mechanical stress are the stimuli that collectively drive these cellular responses by activating a cascade of intracellular signaling mechanisms, including tyrosine kinases, calcium (Ca2+), mitogen-activated protein kinase (MAPK), and protein kinase C (PKC), that affect SMC growth, differentiation, and/or matrix protein synthesis.