Smooth muscle-specific expression of SV40 large TAg induces SMC proliferation causing adaptive arterial remodeling

To study the effects of enhanced smooth muscle cell (SMC) proliferation on arterial vessel geometry in the absence of vessel trauma, we developed a transgenic mouse model expressing SV40 large T antigen under control of the 2.3-kb smooth muscle-myosin heavy chain promoter. Transgenic mice studied at ages from 3 to 13 wk showed a 3.2-fold increase in arterial wall SMC density, with 28% of SMC exhibiting proliferative cell nuclear antigen staining, confirming enhanced SMC proliferation, which was accompanied by two- to threefold increases in arterial wall areas (P < 0.05). Remarkably, despite increased vessel wall mass, the lumen area was not compromised, but rather was increased. A tightly conserved linear relationship was found between arterial circumference and wall thickness with slopes of 0.036 for both transgenics (r = 0.93, P < 0.01) and controls (r = 0.77, P < 0.01), suggesting the hypothesis that the conservation of wall stress functions as a primary determinant of adaptive arterial remodeling. This establishes a new model of adaptive vessel remodeling occurring in response to a proliferative input in the absence of mechanical injury or primary flow perturbation.     

Isolation of a morphologically and functionally distinct smooth muscle cell type from the intimal aspect of the normal rat aorta. Evidence for smooth muscle cell heterogeneity

Summary Recent studies indicate that the neointima of injured rat arteries is composed of a subpopulation of smooth muscle cells (SMCs) distinct from medial smooth muscle cells. However, SMC diversity in normal adult aorta has remained elusive. This study characterizes two morphologically and functionally distinct SMC types isolated from different anatomic regions of the normal rat aorta. Rat aortic medial smooth muscle cells (MSMCs) were isolated from the media after removal of the intimal and adventitial cells. Rat aortic intimal smooth muscle cells (ISMCs) were isolated from the intimal aspect of everted rat aortas. The two cell types were characterized morphologically and immunohistochemically and were compared for their capacity to contract collagen gels in response to endothelin-1. MSMCs were spindle-shaped and grew in hills and valleys showing features previously described for vascular SMCs. Conversely, ISMCs displayed a polygonal and epithelioid shape, grew mainly as a monolayer, and had a higher proliferative rate. Both cell types expressed alpha-smooth muscle actin and were negative for Factor VIII-RAg. ISMCs produced large amounts of a laminin and type IV collagen-rich extracellular matrix which had a characteristic pericellular distribution. ISMCs, but not MSMCs, rapidly contracted collagen gels in response to endothelin-1. This study indicates that the normal rat aorta contains two types of SMCs located in anatomically distinct regions of the vessel wall. Because of their functional characteristics, the SMCs isolated from the intimal aspect of the aorta may play an important role in physiologic as well as pathologic conditions.     

Vascular smooth cell proliferation in perfusion culture of porcine carotid arteries

Objective of this study was to develop a novel in vitro artery culture system to study vascular smooth muscle cell (SMC) proliferation of porcine carotid arteries in response to injury, basic fibroblast growth factor (FGF2), and FGF2 conjugated with cytotoxin saporin (SAP). Perfusion-cultured porcine carotid arteries remained contractile in response to norepinephrine and relaxant to acetylcholine for up to 96 h. SMC proliferation of cultured arteries was detected by bromodeoxyuridine incorporation in both non-injured and balloon-injured arteries. In the inner layer of the vessel wall near the lumen, SMC proliferation were less than 10% in uninjured vessels, 66% in injured vessels, 80% in injured vessels with FGF2 treatment, and 5% in injured vessels with treatment of FGF2-SAP. Thus, the cultured porcine carotid arteries were viable; and the injury stimulated SMC proliferation, which was significantly enhanced by FGF2 and inhibited by FGF2-SAP.     

Generation of Functional Blood Vessels from a Single c-kit+ Adult Vascular Endothelial Stem Cell

In adults, the growth of blood vessels, a process known as angiogenesis, is essential for organ growth and repair. In many disorders including cancer, angiogenesis becomes excessive. The cellular origin of new vascular endothelial cells (ECs) during blood vessel growth in angiogenic situations has remained unknown. Here, we provide evidence for adult vascular endothelial stem cells (VESCs) that reside in the blood vessel wall endothelium. VESCs constitute a small subpopulation within CD117+ (c-kit+) ECs capable of undergoing clonal expansion while other ECs have a very limited proliferative capacity. Isolated VESCs can produce tens of millions of endothelial daughter cells in vitro. A single transplanted c-kit-expressing VESC by the phenotype lin−CD31+CD105+Sca1+CD117+ can generate in vivo functional blood vessels that connect to host circulation. VESCs also have long-term self-renewal capacity, a defining functional property of adult stem cells. To provide functional verification on the role of c-kit in VESCs, we show that a genetic deficit in endothelial c-kit expression markedly decreases total colony-forming VESCs. In vivo, c-kit expression deficit resulted in impaired EC proliferation and angiogenesis and retardation of tumor growth. Isolated VESCs could be used in cell-based therapies for cardiovascular repair to restore tissue vascularization after ischemic events. VESCs also provide a novel cellular target to block pathological angiogenesis and cancer growth.     

Distribution of connexin37, connexin40 and connexin43 in the aorta and coronary artery of several mammals

Intercellular communication between cells of the vessel wall is established by a combination of diffusion and convection of humoral and endothelial factors in the extracellular fluid or by direct intercellular contacts present in the form of gap junctions composed of proteins called connexins. At least connexin (Cx)37, Cx40 and Cx43 are expressed in the vessel wall, but disparate findings with regard to the cell specific localisation of connexins in the vasculature indicate that the distribution of connexins may be species and vessel specific. Moreover, differences in expression exist between cells in culture and tissue sections. We performed an inventory immunohistochemical study on the localisation of Cx37, Cx40 and Cx43 on tissue sections of the bovine, micropig and rat aorta and coronary system, which represent morphologically and functionally different types of vessels in the arterial system. We could observe Cx40 labelling most commonly, although with various intensities, between endothelial and smooth muscle cells of the species studied, with the exception of rat aortic smooth muscle cells. The distribution of Cx43 is more differentiated and mostly confined to smooth muscle cells, although it can be detected scarcely between endothelial cells. Cx37, when detectable, is predominantly expressed between endothelial cells in a heterogeneous pattern. We conclude that Cx40 is the constitutive vascular gap junction protein in situ and guarantees cell coupling between cells in the vessel wall. The differentiated distribution of both Cx37 and Cx43 suggests they are involved in more dynamic processes. Accepted: 12 October 1999     

Development of a general method for designing microvascular networks using distribution of wall shear stress

In the present study, theoretical formulations for calculation of optimal bifurcation angle and relationship between the diameters of mother and daughter vessels using the power law model for non-Newtonian fluids are developed. The method is based on the distribution of wall shear stress in the mother and daughter vessels. Also, the effect of distribution of wall shear stress on the minimization of energy loss and flow resistance is considered. It is shown that constant wall shear stress in the mother and daughter vessels provides the minimum flow resistance and energy loss of biological flows. Moreover, the effects of different wall shear stresses in the mother and daughter branches, different lengths of daughter branches in the asymmetric bifurcations and non-Newtonian effect of biological fluid flows on the bifurcation angle and the relationship between the diameters of mother and daughter branches are considered. Using numerical simulations for non-Newtonian models such as power law and Carreau models, the effects of optimal bifurcation angle on the pressure drop and flow resistance of blood flow in the symmetric bifurcation are investigated. Numerical simulations show that optimal bifurcation angle decreases the pressure drop and flow resistance especially for bifurcations at large Reynolds number.     

Towards understanding of the complex structure of growing yeast populations

In a growing Saccharomyces cerevisiae population, cell size is finely modulated according to both the chronological and genealogical ages. This generates the complex heterogeneous structure typical of budding yeast populations. In recent years, there has been a growing interest in developing mathematical models capable of faithfully describing population dynamics at the single cell level. A multistaged morphologically structured model has been lately proposed based on the population balance theory. The model was able to describe the dynamics of the generation of a heterogeneous growing yeast population starting from a sub-population of daughter unbudded cells. In this work, which aims at validating the model, the simulated experiment was performed by following the release of a homogeneous population of daughter unbudded cells. A biparametric flow cytometric approach allowed us to analyse the time course joint distribution of DNA and protein contents at the single cell level; this gave insights into the coupling between growth and cell cycle progression that generated the final population structure. The comparison between experimental and simulated size distributions revealed a strong agreement for some unexpected features as well. Therefore, the model can be considered as validated and extendable to more complex situations.     

Analysis of cell division by time-lapse cinematographic studies of hydrocortisone-treated embryonic lung fibroblasts

Hydrocortisone is a modulator of cell division and has been shown to prolong the replicative in vitro life span of human embryonic lung fibroblasts. Time lapse cinematography was used to analyze the proliferative behavior of individual cells in populations of fibroblasts exposed to hydrocortisone in young cultures during a single growth cycle and in aged cultures that had been continuously exposed to hydrocortisone. Results indicate that hydrocortisone causes a decrease in the interdivision time (IDT) of a portion of the cells in the population and this effect is augmented after continuous exposure to hydrocortisone. Hydrocortisone does not appear to increase the number of initial dividers in the population but increases growth rate in the early stages of the culture period. Analysis of mother-daughter IDT pairs further suggests that hydrocortisone exerts its effects on IDT independently for a given cell.     

毛乌素沙地根茎禾草拂子茅对异质性水分供应的表型可塑性

拂子茅(Calamagrostis epigejos（L.)Roth)为根茎型多年生禾草,具细长根茎。为了探讨拂子茅在异质性水分环境中的表型差异,在内蒙古鄂尔多斯高原的毛乌素沙地对拂子茅由母株、子株组成的分株对给予了高水、低水两种不同的异质性土壤水分处理。实验结果表明：土壤水分状况显著地影响着拂子茅分株的生长表型。在高土壤水分条件下,拂子茅的分株产生的根茎、新生后代分株较多,并使生物量主要分配于地上部分,地上生物量积累多;在低土壤水分条件下,拂子茅分株产生较少的根茎与新生后代分株,并且分配到根系的生物量明显增大。在具有一定对比度的异质性土壤水分环境中,拂子茅分株并不因相连的其他分株所处的土壤水分状况而在根茎生长、新生后代分株的产生和生物量分配等特征上,与同质环境中的具有相同土壤水分状况的分株相比,有明显差异。这些结果揭示：拂子茅仅以分株的形式对异质性水分供应发生表型反应;相连的克隆分株在向顶向和向基向这两个基本方向上,不能对另一分株的土壤水分状况在生K表型上发生反应,它们在水分关系上可能是相互相对独立的。分株的相对独立可能有利于在气候干旱、扰动强烈的沙地环境中实现风险分摊,提高基株的存活几率。     

Blood vessel maturation in a 3-dimensional spheroidal coculture model: direct contact with smooth muscle cells regulates endothelial cell quiescence and abrogates VEGF responsiveness.

Paracrine interactions between endothelial cells (EC) and mural cells act as critical regulators of vessel wall assembly, vessel maturation and define a plasticity window for vascular remodeling. The present study was aimed at studying blood vessel maturation processes in a novel 3-dimensional spheroidal coculture system of EC and smooth muscle cells (SMC). Coculture spheroids differentiate spontaneously in a calcium-dependent manner to organize into a core of SMC and a surface layer of EC, thus mimicking the physiological assembly of blood vessels with surface lining EC and underlying mural cells. Coculture of EC with SMC induces a mature, quiescent EC phenotype as evidenced by 1) a significant increase in the number of junctional complexes of the EC surface layer, 2) a down-regulation of PDGF-B expression by cocultured EC, and 3) an increased resistance of EC to undergo apoptosis. Furthermore, EC cocultured with SMC become refractory to stimulation with VEGF (lack of CD34 expression on VEGF stimulation; inability to form capillary-like sprouts in a VEGF-dependent manner in a 3-dimensional in gel angiogenesis assay). In contrast, costimulation with VEGF and Ang-2 induced sprouting angiogenesis originating from coculture spheroids consistent with a model of Ang-2-mediated vessel destabilization resulting in VEGF responsiveness. Ang-2 on its own was able to stimulate endothelial cells in the absence of Ang-1 producing SMC, inducing lateral sheet migration as well as in gel sprouting angiogenesis. Taken together, the data establish the spheroidal EC/SMC system as a powerful cell culture model to study paracrine interactions in the vessel wall and provide functional evidence for smooth muscle cell-mediated quiescence effects on endothelial cells.     

毛乌素沙地根茎禾草拂子茅对异质性水分供应的表型可塑性

拂子茅(Calamagrostis epigejos(L.)Roth.)为根茎型多年生禾草,具细长根茎.为了探讨拂子茅在异质性水分环境中的表型差异,在内蒙古鄂尔多斯高原的毛乌素沙地对拂子茅由母株、子株组成的分株对给予了高水、低水两种不同的异质性土壤水分处理.实验结果表明:土壤水分状况显著地影响着拂子茅分株的生长表型.在高土壤水分条件下,拂子茅的分株产生的根茎、新生后代分株较多,并使生物量主要分配于地上部分,地上生物量积累多;在低土壤水分条件下,拂子茅分株产生较少的根茎与新生后代分株,并且分配到根系的生物量明显增大.在具有一定对比度的异质性土壤水分环境中,拂子茅分株并不因相连的其他分株所处的土壤水分状况而在根茎生长、新生后代分株的产生和生物量分配等特征上,与同质环境中的具有相同土壤水分状况的分株相比,有明显差异.这些结果揭示:拂子茅仅以分株的形式对异质性水分供应发生表型反应;相连的克隆分株在向顶向和向基向这两个基本方向上,不能对另一分株的土壤水分状况在生长表型上发生反应,它们在水分关系上可能是相互相对独立的.分株的相对独立可能有利于在气候干旱、扰动强烈的沙地环境中实现风险分摊,提高基株的存活几率.     

Influence of tensile strain on smooth muscle cell orientation in rat blood vessels.

Blood vessels are subject to tensile stress and associated strain which may influence the structure and organization of smooth muscle cells (SMCs) during physiological development and pathological remodeling. This study focused on the influence of the major tensile strain on the SMC orientation in the blood vessel wall. Several blood vessels, including the aorta, the mesenteric artery and vein, and the jugular vein of the rat were used to observe the normal distribution of tensile strains and SMC orientation; and a vein graft model was used to observe the influence of altered strain direction on the SMC orientation. The circumferential and longitudinal strains in these blood vessels were measured by using a biomechanical technique, and the SMC orientation was examined by fluorescent microscopy at times of 10, 20, and 30 days. Results showed that the SMCs were mainly oriented in the circumferential direction of straight blood vessels with an average angle of approximately 85 deg between the SMC axis and the vessel axis in all observed cases. The SMC orientation coincided with the principal direction of the circumferential strain, a major tensile strain, in the blood vessel wall. In vein grafts, the major tensile strain direction changed from the circumferential to the longitudinal direction at observation times of 10, 20, and 30 days after graft surgery. This change was associated with a decrease in the angle between the axis of newly proliferated SMCs and that of the vessel at all observation times (43 +/- 11 deg, 42 +/- 10 deg, and 41 +/- 10 deg for days 10, 20, and 30, respectively), indicating a shift of the SMC orientation from the circumferential toward the longitudinal direction. These results suggested that the major tensile strain might play a role in the regulation of SMC orientation during the development of normal blood vessels as well as during remodeling of vein grafts.     

Smooth muscle-endothelial cell communication activates Reelin signaling and regulates lymphatic vessel formation

Active lymph transport relies on smooth muscle cell (SMC) contractions around collecting lymphatic vessels, yet regulation of lymphatic vessel wall assembly and lymphatic pumping are poorly understood. Here, we identify Reelin, an extracellular matrix glycoprotein previously implicated in central nervous system development, as an important regulator of lymphatic vascular development. Reelin-deficient mice showed abnormal collecting lymphatic vessels, characterized by a reduced number of SMCs, abnormal expression of lymphatic capillary marker lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1), and impaired function. Furthermore, we show that SMC recruitment to lymphatic vessels stimulated release and proteolytic processing of endothelium-derived Reelin. Lymphatic endothelial cells in turn responded to Reelin by up-regulating monocyte chemotactic protein 1 (MCP1) expression, which suggests an autocrine mechanism for Reelin-mediated control of endothelial factor expression upstream of SMC recruitment. These results uncover a mechanism by which Reelin signaling is activated by communication between the two cell types of the collecting lymphatic vessels--smooth muscle and endothelial cells--and highlight a hitherto unrecognized and important function for SMCs in lymphatic vessel morphogenesis and function.     

Microtubule organization during development of stomatal complexes inLolium rigidum

Summary Microtubule (MT) arrays in stomatal complexes ofLolium have been studied using cryosectioning and immunofluorescence microscopy. This in situ analysis reveals that the arrangement of MTs in pairs of guard cells (GCs) or subsidiary cells (SCs) within a complex is very similar, indicating that MT deployment is closely coordinated during development. In premitotic guard mother cells (GMCs), MTs of the transverse interphase MT band (IMB) are reorganized into a longitudinal array via a transitory array in which the MTs appear to radiate from the cell edges towards the centre of the walls. Following the longitudinal division of GMCs, cortical MTs are reinstated in the GCs at the edge of the periclinal and ventral walls. The MTs become organized into arrays which radiate across the periclinal walls, initially from along the length of the ventral wall and later only from the pore site. As the GCs elongate, the organization of MTs and the patterns of wall expansion differ on the internal and external periclinal walls. A final reorientation of MTs from transverse to longitudinal is associated with the elongation and constriction of GCs to produce mature complexes. During cytokinesis in the subsidiary mother cells (SMCs), MTs appear around the reforming nucleus in the daughter epidermal cells but appear in the cortex of the SC once division is complete. Our results are thus consistent with the idea that interphase MTs are nucleated in the cell cortex in all cells of the stomatal complex but not in adjacent epidermal cells.Abbreviations GMC guard mother cell - GC guard cell - IMB interphase microtubule band - MT microtubule - PPB preprophase band - SMC subsidiary mother cell - SC subsidiary cell     

Physiological cyclic stretch causes cell cycle arrest in cultured vascular smooth muscle cells

Smooth muscle cells (SMC) are the major cellular component of the blood vessel wall and are continuously exposed to cyclic stretch due to pulsatile blood flow. This study examined the effects of a physiologically relevant level of cyclic stretch on rat aortic vascular SMC proliferation. Treatment of static SMC with serum, platelet-derived growth factor, or thrombin stimulated SMC proliferation, whereas exposure of SMC to cyclic stretch blocked the proliferative effect of these growth factors. The stretch-mediated inhibition in SMC growth was not due to cell detachment or increased cell death. Flow cytometry analysis revealed that cyclic stretch increased the fraction of SMC in the G(0)/G(1) phase of the cell cycle. Stretch-inhibited G(1)/S phase transition was associated with a decrease in retinoblastoma protein phosphorylation and with a selective increase in the cyclin-dependent kinase inhibitor p21, but not p27. These results demonstrate that cyclic stretch inhibits SMC growth by blocking cell cycle progression and suggest that physiological levels of cyclic stretch contribute to vascular homeostasis by inhibiting the proliferative pathway of SMC.     

Multiple repressor pathways contribute to phenotypic switching of vascular smooth muscle cells

Smooth muscle cell (SMC) differentiation is an essential component of vascular development and these cells perform biosynthetic, proliferative, and contractile roles in the vessel wall. SMCs are not terminally differentiated and possess the ability to modulate their phenotype in response to changing local environmental cues. The focus of this review is to provide an overview of the current state of knowledge of molecular mechanisms involved in controlling phenotypic switching of SMC with particular focus on examination of processes that contribute to the repression of SMC marker genes. We discuss the environmental cues which actively regulate SMC phenotypic switching, such as platelet-derived growth factor-BB, as well as several important regulatory mechanisms required for suppressing expression of SMC-specific/selective marker genes in vivo, including those dependent on conserved G/C-repressive elements, and/or highly conserved degenerate CArG elements found in the promoters of many of these marker genes. Finally, we present evidence indicating that SMC phenotypic switching involves multiple active repressor pathways, including Krüppel-like zinc finger type 4, HERP, and ERK-dependent phosphorylation of Elk-1 that act in a complementary fashion. serum response factor; platelet-derived growth factor-BB     

Distribution and function of actin in the developing stomatal complex of winter rye (Secale cereale cv. Puma)

Summary The dynamics of actin distribution during stomatal complex formation in leaves of winter rye was examined by means of immunofluorescence microscopy of epidermal sheets. This method results in actin localization patterns that are the same as those seen with rhodamine-phalloidin staining, but are more stable. During stomatal development MFs are extensively rearranged, and most of the time the orientation or placement of MFs is distinctly different from that of MTs, the exception being co-localization of MTs and MFs in phragmoplasts. Although MFs show an orientation similar to that of MTs in interphase guard mother cells, no banding of MFs into anything resembling the interphase MT band is observed. From prophase to telophase, a distinct, dense concentration of MFs is found in subsidiary cell mother cells (SMCs) between the nucleus and the region of the cell cortex facing the guard mother cell. Cytochalasin B treatment causes incorrect positioning of the SMC nucleus/daughter nuclei and abarrent placement and orientation of the new cell wall that forms the boundary of the subsidiary cell at cytokinesis. These results suggest that MFs are involved in maintaining the SMC nucleus in its correct position and the SMC spindle in the correct orientation relative to the division site previously delineated by the preprophase band. Because these MFs thus appear to assure that the SMC phragmoplast begins to form in the correct orientation near the division site to which it needs to grow, we suggest that MFs are involved in control of correct placement and orientation of the new cell wall of the subsidiary cell.Abbreviations CB cytochalasin B - DIC differential interference contrast - DMSO dimethylsulfoxide - MBS m-maleimidobenzoyl-N-hydroxylsuccinimide ester - MF microfilament - MT microtubule - PBS phosphate buffered saline - SMC subsidiary cell mother cell Dedicated to the memory of Professor Oswald Kiermayer     

Numerical investigation of the non-Newtonian pulsatile blood flow in a bifurcation model with a non-planar branch

The pulsatile flow of non-Newtonian fluid in a bifurcation model with a non-planar daughter branch is investigated numerically by using the Carreau-Yasuda model to take into account the shear thinning behavior of the analog blood fluid. The objective of this study is to deal with the influence of the non-Newtonian property of fluid and of out-of-plane curvature in the non-planar daughter vessel on wall shear stress (WSS), oscillatory shear index (OSI), and flow phenomena during the pulse cycle. The non-Newtonian property in the daughter vessels induces a flattened axial velocity profile due to its shear thinning behavior. The non-planarity deflects flow from the inner wall of the vessel to the outer wall and changes the distribution of WSS along the vessel, in particular in systole phase. Downstream of the bifurcation, the velocity profiles are shifted toward the flow divider, and low WSS and high shear stress temporal oscillations characterized by OSI occur on the outer wall region of the daughter vessels close to the bifurcation. Secondary motions become stronger with the addition of the out-of-plane curvature induced by the bending of the vessel, and the secondary flow patterns swirl along the non-planar daughter vessel. A significant difference between the non-Newtonian and the Newtonian pulsatile flow is revealed during the pulse cycle; however, reasonable agreement between the non-Newtonian and the rescaled Newtonian flow is found. Calculated results for the pulsatile flow support the view that the non-planarity of blood vessels and the non-Newtonian properties of blood are an important factor in hemodynamics and may play a significant role in vascular biology and pathophysiology.