Sex steroid-mediated reprogramming of vascular smooth muscle cells to stem cells and neurons: Possible utilization of sex steroid combinations for regenerative treatment without utilization of in vitro developed stem cells

Previous work from our laboratory demonstrated that sex steroid combinations, but not individual sex steroids alone, cause transdifferentiation of ovarian epithelial cells - ovarian surface epithelium (OSE) and follicular granulosa cells - into neural stem cells (NSC) and differentiating neurons. In the present study we have chosen primary culture of human vascular smooth muscle cells (SMC), a non-epithelial mesenchymal cells in order to test them as a control cell type regarding their morphology and expression of NSC and neuronal markers. Utilization of estradiol (E2), progesterone (PG) or testosterone (TS) alone did not induce the emergence of neurons from the vascular SMC. However, the treatment with sex steroid combinations (PG+TS or E2+PG+TS) caused transdifferentiation into neural/neuronal type cells. By immunohistochemistry, these cells exhibited strong expression of stem cell markers and neural/neuronal glycoconjugates SSEA-1, SSEA-4, Thy-1, NeuN and NCAM. In the Neurobasal/B27 medium both, the OSE and vascular SMC also transdifferentiated into neuronal cells. Western blot analysis has shown significant increase of NeuN 48-kDa species after E2+PG or PG+TS treatment. Secretion of E2 increased significantly in vascular SMC cultures pretreated with TS, PG or TS+PG. Unlike OSE cells, the vascular SMC accompany as pericytes all vessels, including CNS microvasculature. We also observed that sex steroid combinations could produce SMC stem type cells which differentiated within a few days back to mature vascular SMC. This is of potential interest for the vascular regenerative medicine. Altogether, our observations suggest that sex steroid combinations could induce in vivo improvement of neurodegenerative, traumatic and ischemic neurological disorders and vascular diseases via their effect on resident pluripotent vascular SMC, i.e. without a need of in vitro developed stem cells.     

Fluid shear stress-induced alignment of cultured vascular smooth muscle cells

The study objectives were to quantify the time- and magnitude-dependence of flow-induced alignment in vascular smooth muscle cells (SMC) and to identify pathways related to the orientation process. Using an intensity gradient method, we demonstrated that SMC aligned in the direction perpendicular to applied shear stress, which contrasts with parallel alignment of endothelial cells under flow SMC alignment varied with the magnitude of and exposure time to shear stress and is a continuous process that is dependent on calcium and cycloskeleton based mechanisms. A clear understanding and control of flow-induced SMC alignment will have implications for vascular tissue engineering.     

Vascular beta-adrenoceptor function in hypertension and in ageing

Functional beta-adrenoceptors (beta-AR) have been identified and characterized in blood vessels under in vivo conditions as well as in vascular smooth muscle cells (SMC) grown in culture. Agonist occupancy of beta-AR activates adenylyl cyclase (AC) via the stimulatory guanine nucleotide-binding protein (Gs) and leads to elevations in intracellular adenosine 3'5'-cyclic monophosphate levels (cAMP). Increased cAMP activates the cAMP-dependent protein kinase (PKA), with subsequent phosphorylation of various target proteins. This beta-AR pathway interacts with several other intracellular signalling pathways via cross-talk, so that activation by beta-AR agonists may also modulate other second messengers and protein kinases. SMC beta-AR play an important role in SMC function. In intact blood vessels they mediate SMC relaxation by various intracellular mechanisms, ultimately causing a decrease in intracellular Ca2+ levels. In cultured SMC, activation of the beta-AR pathway results in inhibition of cellular proliferation, the development of SMC polyploidy, and SMC apoptosis. Blood vessels from hypertensive animals are characterized by an increase in SMC cell mass, a greater incidence of SMC polyploidy in the aorta, and an impairment in the beta-agonist-mediated SMC relaxation. Some of these changes may result from an attenuation of beta-AR function due to agonist-induced receptor desensitization caused by the uncoupling of receptors from the Gs-AC system. The phosphorylated beta-AR may in turn trigger new signals and activate different intracellular pathways. However, the details of these mechanisms are still unresolved. Since functional beta-AR play such a prominent and multi-faceted role in SMC function, it is important to understand how these diverse physiological effects are mediated by this receptor system, and how they contribute to the development of hypertension. With ageing, a decrease in beta-AR-Gs-AC coupling is observed, and this is implicated in the reduced responsiveness of SMC. The similarities in SMC beta-AR functional changes in hypertension and in ageing suggest that the underlying mechanisms are also analogous.     

Evidence for cytokine regulation of cholesterol metabolism in herpesvirus-infected arterial cells by the lipoxygenase pathway

Cytokines such as tumor necrosis factor (TNF), interleukin-1 (IL-1), and gamma-interferon (IF) are produced by activated hematopoietic cells. They possess antiviral activity and have other biological activities such as induction of cell proliferation and hemorrhagic necrosis of tumors. Since herpes simplex virus (HSV) infection of human vascular cells is known to produce a biochemical and cytopathological effect virtually indistinguishable from atherosclerosis, we hypothesized that these cytokines many prevent cholesteryl ester (CE) accumulation in arterial smooth muscle cells (SMC) that is seen with herpesvirus infection. We now report that TNF and IL-1 but not gamma-IF prevent CE accumulation in HSV-infected arterial SMC by induction of cyclic AMP-dependent CE hydrolysis. This effect is mediated through the arachidonate 12-lipoxygenase pathway via 12-HETE since pretreatment of cells with several lipoxygenase inhibitors abolishes the antiviral effect and 12-HETE is the major (greater than 99%) lipoxygenase metabolite produced by these cells. This conclusion is further based on our observations that TNF and IL-1 enhance 12-HETE production in SMC and that 12-HETE significantly increases both intracellular cyclic AMP and lysosomal CE hydrolysis. Moreover, dibutyryl cyclic AMP restored a normal phenotype in these virally infected cells. Collectively, these findings identify for the first time a biochemical mechanism involved in the reduction of lipid accumulation in virally infected arterial SMC by these potent cytokines.     

Functional significance of human vascular smooth muscle cell-derived interleukin 1 in paracrine and autocrine regulation pathways.

Interleukin 1 (IL1), a key mediator in the cytokine network, alters many pathophysiologically important functions of blood vessel wall cells. Vascular cells, such as endothelial cells and smooth muscle cells (SMC) can themselves transcribe IL1 genes, raising the possibility that IL1 regulates blood vessel wall functions by local autocrine or paracrine mechanisms. However, IL1 lacks a recognizable signal sequence and it is still unclear how vascular cells might release IL1 or if IL1 derived from vascular cells can actually produce autocrine or paracrine effects. We explored these issues in human vascular SMC, the most numerous cell type in arteries and veins, using cultured SMC and short term organoid cultures. SMC treated with lipopolysaccharide recombinant tumor necrosis factor (recTNF), or recIL1 itself ("activated SMC") elaborated thymocyte costimulatory activity, a biological activity traditionally ascribed to IL1. However, neutralization experiments with monospecific antibodies disclosed that the more recently recognized cytokine IL6 rather than IL1 accounted for most of the soluble thymocyte costimulatory activity released by activated SMC. Using the D10S assay that distinguishes IL1 from IL6 and TNF we found that the culture supernatant of activated SMC contained little or no IL1, but that the cytosol and surface of these cells did exhibit this activity. Antiserum to recIL1 alpha inhibited stimulation of D10S cells by surface-associated IL1 of activated SMC, while treatment with acid to elute receptor- or nonspecifically bound IL1 did not abrogate this D10S proliferation. Short term organoid cultures of both normal veins and human arteriosclerotic plaque also expressed tissue-associated IL1 activity upon stimulation with LPS but did not release significant soluble IL1 activity. To establish further the biological functions of cell-associated IL1, we incubated stimulated or unstimulated SMC that were fixed with paraformaldehyde and washed extensively (fixed SMC) with overlayered viable SMC of the same donor (responder SMC). Contact with fixed SMC that bore surface IL1 following TNF or IL1 stimulation evoked up to 20-fold higher IL6 release from responder SMC than did exposure to unstimulated SMC (57 vs 1052 ng/ml/day). Addition of anti-IL1 antibody inhibited the release of IL6 from the responder SMC. These results demonstrate that cytokine-activated SMC express biologically active IL1 on their cell surface and illustrate how these cells might actually participate in autocrine and paracrine signaling in the vessel wall. The requirement for direct intercellular contact for IL1 effects could facilitate local information exchange among vascular wall cells and/or infiltrating leukocytes and permit costimulation while limiting undue propagation of inflammatory stimuli.     

Beta-type transforming growth factor specifies organizational behavior in vascular smooth muscle cell cultures

In culture, vascular smooth muscle cells (SMC) grow in a "hill-and-valley" (multilayered) pattern of organization. We have studied the growth, behavioral organization, and biosynthetic phenotype of rat aortic SMC exposed to purified platelet-derived growth regulatory molecules. We show that multilayered growth is not a constitutive feature of cultured SMC, and that beta-type transforming growth factor (TGF-beta) is the primary determinant of multilayered growth and the hill-and-valley pattern of organization diagnostic for SMC in culture. TGF-beta inhibited, in a dose-dependent manner, the serum- or platelet-derived growth factor-mediated proliferation of these cells in two-dimensional culture, but only when cells were plated at subconfluent densities. The ability of TGF-beta to inhibit SMC growth was inversely correlated to plating cell density. When SMC were plated at monolayer density (5 X 10(4) cells/cm2) to allow maximal cell-to-cell contact, TGF-beta potentiated cell growth. This differential response of SMC to TGF-beta may contribute to the hill-and-valley pattern of organization. Unlike its effect on other cell types, TGF-beta did not enhance the synthesis of fibronectin or its incorporation into the extracellular matrix. However, the synthesis of a number of other secreted proteins was altered by TGF-beta treatment. SMC treated with TGF-beta for 4 or 8 h secreted markedly enhanced amounts of an Mr 38,000-D protein doublet whose synthesis is known to be increased by heparin (another inhibitor of SMC growth), suggesting metabolic similarities between heparin- and TGF-beta-mediated SMC growth inhibition. The data suggest that TGF-beta may play an important and complex regulatory role in SMC proliferation and organization during development and after vascular injury.     

Endothelium-independent conversion of angiotensin I by vascular smooth muscle cells

The conversion of angiotensin I (AT-I) to angiotensin II (AT-II) by angiotensin I-converting enzyme (ACE) is a key step in the action of angiotensins. ACE is constitutively expressed in endothelial cells, but can also be detected at low levels in smooth muscle cells (SMC). Furthermore, in rats the ACE activity can be induced in SMC in vivo by experimental hypertension or vascular injury and in vivo by corticoid treatment. This study was therefore undertaken to evaluate the conversion of AT-I and its subsequent effects in SMC in basal conditions and after stimulation by dexamethasone. Using rat and human SMC, showed that dexamethasone induced ACE expression and that this enzyme was functional, leading to AT-II-dependent intracellular signaling. A fourfold increase in phospholipase C activity in response to AT-I was observed in dexamethasone-activated SMC compared with quiescent SMC. This effect of dexamethasone on signal transduction is dependent on ACE activity, whereas AT-II receptor parameters remain unchanged. The action of AT-I was blocked by an AT1 receptor antagonist, suggesting that it was mediated by AT-II. Similarly, dexamethasone-induced ACE expression was present in human SMC, and calcium signaling was mobilized in response to AT-I in activated human cells. Experiments performed with cocultures of endothelial cells and SMC in a Transwell system showed that the response to AT-I was limited to the compartment where AT-I was localized, suggesting that AT-I does not pass through the endothelial cell barrier to interact with underlying SMC. Our data suggest that in rat, as in human SMC, the conversion of AT-I into AT-II and the signal transduction in response to AT-I are ACE expression-dependent. In addition, the present findings show that this SMC response to AT-I is endothelium-independent, supporting the idea of a local generation of AT-II in the vascular wall.     

Disruption of TGF-β signaling in smooth muscle cell prevents flow-induced vascular remodeling

Transforming growth factor-β (TGF-β) signaling has been prominently implicated in the pathogenesis of vascular remodeling, especially the initiation and progression of flow-induced vascular remodeling. Smooth muscle cells (SMCs) are the principal resident cells in arterial wall and are critical for arterial remodeling. However, the role of TGF-β signaling in SMC for flow-induced vascular remodeling remains unknown. Therefore, the goal of our study was to determine the effect of TGF-β pathway in SMC for vascular remodeling, by using a genetical smooth muscle-specific (SM-specific) TGF-β type II receptor (Tgfbr2) deletion mice model. Mice deficient in the expression of Tgfbr2 (MyhCre.Tgfbr2^f/f) and their corresponding wild-type background mice (MyhCre.Tgfbr2^WT/WT) underwent partial ligation of left common carotid artery for 1, 2, or 4 weeks. Then the carotid arteries were harvested and indicated that the disruption of Tgfbr2 in SMC provided prominent inhibition of vascular remodeling. And the thickening of carotid media, proliferation of SMC, infiltration of macrophage, and expression of matrix metalloproteinase (MMP) were all significantly attenuated in Tgfbr2 disruption mice. Our study demonstrated, for the first time, that the TGF-β signaling in SMC plays an essential role in flow-induced vascular remodeling and disruption can prevent this process.     

ANG II type 2 receptor regulates smooth muscle growth and force generation in late fetal mouse development

Although evidence from culture studies implicates the angiotensin II (ANG II) type 2 receptor (AT(2)R) in the regulation of growth and differentiation of arterial smooth muscle (SM) cells (SMC), the lack of its expression in adult arteries has precluded direct investigation of its role in vivo. The goal of the present study was to determine the role of AT(2)R in the control of fetal SMC growth, contractility, and differentiation during vascular development. Determination of isometric tension in fetal aortas showed potentiated ANG II-induced contraction by treatment with the selective AT(2)R antagonist PD-123319, demonstrating the presence of functional AT(2)Rs that mediate reduced force development in vascular SMC. In direct contrast to numerous cell culture studies, proliferation indexes were decreased rather than increased in aortic SMC of fetal homozygous AT(2)R knockout compared with wild-type or heterozygous knockout mice. Experiments using SMC tissues from heterozygous female AT(2)R knockout mice, which are naturally occurring chimeras for AT(2)R expression, showed that AT(2)R mRNA expression was exactly 50% of that of wild type. This indicated that loss of AT(2)R expression did not confer a selective advantage or disadvantage for SMC lineage determination and expansion. Real time RT-PCR analyses showed no significant difference in expression of SM-alpha-actin, SM myosin heavy chain, and myocardin in various SM tissues from all three genotypes, suggesting that knockout of AT(2)R had no effect on subsequent SMC differentiation. Taken together, results indicate that functional AT(2)R are expressed in fetal aorta and mediate reduced force development but do not significantly contribute to regulation of SMC differentiation.     

Strain and site dependence of polyploidization of cultured rat smooth muscle

Smooth muscle cell (SMC) growth may play an important role in the pathogenesis of vascular diseases such as atherosclerosis and hypertension. Recent studies have demonstrated that, under different growth stimuli in vivo, SMC may respond by proliferation of diploid cells, polyploidization to the tetraploid (or even octaploid) state, or both. In this study, we used flow cytometry to evaluate the intrinsic tendencies of aortic SMC and nonarterial cells from rats of different strains, ages, and blood pressures to polyploidize in response to in vitro growth stimulation. Significant strain-related differences in polyploidization of aortic SMC were found (P less than 0.001): highest in WKY (normotensive inbred rat related to SHR), intermediate in SHR (genetically hypertensive rat), and lowest in Sprague-Dawley and Fischer (normotensive outbred and inbred rats). Animal age had less or no effect on the degree of polyploidization. Nonarterial cells (venous SMC and lung cells) from WKY and SHR remained essentially diploid, suggesting tissue specificity of in vitro polyploidization. Studies of the growth kinetics of uncloned and clonal populations of aortic SMC revealed decreased proliferation as the ploidy increased in WKY, SHR, and Sprague-Dawley. These findings suggest that genetic strain factors as well as cell type/site of origin significantly influence in vitro polyploidization, whereas animal age and blood pressure do not. The findings also emphasize the need to consider ploidy changes when evaluating in vitro SMC growth kinetics. Further studies will improve understanding of SMC growth regulation and the functional significance of vascular polyploidy.     

Host bone-marrow cells are a source of donor intimal smooth- muscle-like cells in murine aortic transplant arteriopathy

Long-term solid-organ allografts typically develop diffuse arterial intimal lesions (graft arterial disease; GAD), consisting of smooth-muscle cells (SMC), extracellular matrix and admixed mononuclear leukocytes. GAD eventually culminates in vascular stenosis and ischemic graft failure. Although the exact mechanisms are unknown, chronic low-level alloresponses likely induce inflammatory cells and/or dysfunctional vascular wall cells to secrete growth factors that promote SMC intimal recruitment, proliferation and matrix synthesis. Although prior work demonstrated that the endothelium and medial SMCs lining GAD lesions in cardiac allografts are donor-derived, the intimal SMC origin could not be determined. They are generally presumed to originate from the donor media, leading to interventions that target donor medial SMC proliferation, with limited efficacy. However, other reports indicate that allograft vessels may contain host-derived endothelium and SMCs (refs. 8,9). Moreover, subpopulations of bone-marrow and circulating cells can differentiate into endothelium, and implanted synthetic vascular grafts are seeded by host SMCs and endothelium. Here we used murine aortic transplants to formally identify the source of SMCs in GAD lesions. Allografts in beta-galactosidase transgenic recipients showed that intimal SMCs derived almost exclusively from host cells. Bone-marrow transplantation of beta-galactosidase--expressing cells into aortic allograft recipients demonstrated that intimal cells included those of marrow origin. Thus, smooth-muscle--like cells in GAD lesions can originate from circulating bone--marrow-derived precursors.     

Role for furin in tumor necrosis factor alpha-induced activation of the matrix metalloproteinase/sphingolipid mitogenic pathway

Neutral sphingomyelinase (nSMase), the initial enzyme of the sphingolipid signaling pathway, is thought to play a key role in cellular responses to tumor necrosis factor alpha (TNF-alpha), such as inflammation, proliferation, and apoptosis. The mechanism of TNF-alpha-induced nSMase activation is only partly understood. Using biochemical, molecular, and pharmacological approaches, we found that nSMase activation triggered by TNF-alpha is required for TNF-alpha-induced proliferation and in turn requires a proteolytic cascade involving furin, membrane type 1 matrix metalloproteinase (MT1-MMP), and MMP2, and leading finally to extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation and DNA synthesis, in smooth muscle cells (SMC) and fibroblasts. Pharmacological and molecular inhibitors of MMPs (batimastat), furin (alpha1-PDX inhibitor-transfected SMC), MT1-MMP (SMC overexpressing a catalytically inactive MT1-MMP), MMP2 (fibroblasts from MMP2(-/-) mice), and small interfering RNA (siRNA) strategies (siRNAs targeting furin, MT1-MMP, MMP2, and nSMase) resulted in near-complete inhibition of the activation of nSMase, sphingosine kinase-1, and ERK1/2 and of subsequent DNA synthesis. Exogenous MT1-MMP activated nSMase and SMC proliferation in normal but not in MMP2(-/-) fibroblasts, whereas exogenous MMP2 was active on both normal and MMP2(-/-) fibroblasts. Altogether these findings highlight a pivotal role for furin, MT1-MMP, and MMP2 in TNF-alpha-induced sphingolipid signaling, and they identify this system as a possible target to inhibit SMC proliferation in vascular diseases.     

A new concept of development of neointimal hyperplasia

The intima hyperplasia is a major morphological feature of various arterial pathologies such as atherosclerosis, postangioplasty restenosis and transplantation arteriopathy. It is commonly assumed that smooth muscle cells (SMC) comprising loci of the intima hyperplasia originate from arterial media. However, recent studies suggest that the bone marrow could also supply circulating vascular progenitor of SMCs and endothelial cells (EC). Such bone marrow progenitors participate in the formation of a cellular mass of neointima after experimental allotransplantation, mechanical vessel injury or hyperlipidemia induced experimental atherosclerosis. Circulating SMC and EC progenitors are also likely to be involved in the transplantation arteriopathy development in humans but their roles in the atherosclerosis and restenosis remain to be determined. Stages of the mobilization, defferentiation and proliferation of SMC progenitors could provide point of attack for new therapeutic strategies for the treatment of proliferative vascular diseases. The precise understanding of the neointima cells origin could provide a key for development of the optimal therapeutic strategy of treatnent of such disorders. This review is focused on the pathological significance of circulating progenitors of the bone marrow origin, particularly on the SMC progenitors, for development of vascular wall disorders.