Effects of hypoxia on endothelin-1 sensitivity in the corpus cavernosum

The penis remains in a hypo-oxygenated, flaccid state for a large majority of the time. In this study, we investigated the effect of changing oxygen tension on the expression and functional activity of endothelin-1 (ET-1) receptors in the penis. Experiments were performed in rabbit and human corpora cavernosa (CC) as well as in human fetal penile tissue and cell cultures [human fetal penile endothelial cells (hfPECs) and human fetal smooth muscle cells (hfPSMCs)]. Endothelin A (ETA) receptors are expressed by both endothelial and muscular cells in all tissues investigated. Only penile endothelial cells express endothelin B (ETB) receptors, which are further turned on during experimental hypoxia. In addition, hypoxia also allows ETB expression in the muscular compartment without affecting ETA expression. This hypoxia-induced over-expression of ETB decreased the contractile activity of ET-1 and increased ETB-mediated relaxation. The latter was essentially related to increased ETB-mediated nitric oxide formation in hfPEC and even in hfPSMC. Hypoxia also induced a time-dependent down-regulation of RhoA and Rho kinase (ROK) expression which, in turn, participated in the decreased contractile activity of ET-1 in the hypoxic penile tissue. Accordingly, during hypoxia, an ROK inhibitor, Y27632, was less effective in relaxing ET-1-precontracted strips. In conclusion, prolonged (24 h) hypoxia stimulated several counter-regulatory mechanisms in penile tissue, including up-regulation of ETB and down-regulation of RhoA/ROK pathways, which may help to preserve CC hypo-oxygenation, allowing smooth muscle relaxation and, most probably, penile erection.     

Identification, characterization and biological activity of oxytocin receptor in the developing human penis

Although abnormalities of the male external genitalia (MEG) are a relatively common problem, little is known concerning the molecular mechanisms that finely regulate penile development. We report here the expression of the oxytocin receptor (OTR) gene by real-time RT-PCR in human fetal tissues (11th-12th week of gestation), including the MEG. The developing penis expressed a very high level of OTR mRNA, only a half log(10) unit lower than fetal central nervous system, used as a positive control. The OTR protein is also highly expressed (western, immunohistochemistry and binding studies) and immunolocalized both in the mesenchymal body and in the surrounding blood capillaries, which will later constitute penile trabeculae and sinusoids. Binding studies using [125I]oxytocin antagonist ([125I]OTA) in cultured human fetal penile smooth muscle cells (hfPSMC) revealed the presence of specific OTR with a high capacity and affinity for oxytocin (OT) and for OTA. Increasing concentrations of OT dose-dependently induced intracellular Ca2+ mobilization. Furthermore, OTR mediated an increase in the proliferation and the migration of hfPSMC. In conclusion, we demonstrate that in the developing human MEG, OTR is highly expressed and might be involved in coordinating timely and appropriate proliferation and migration of the penile cells. Thus, OTR might represent an additional target for investigating human fetal MEG organogenesis.     

Regulation of matrix metalloproteinases (MMPs) and their tissue inhibitors in human myometrial smooth muscle cells by TGF-beta1.

The objective of the present study was to determine whether transforming growth factor beta (TGF-beta) regulates the expression of matrix metalloproteinases (MMP) and the tissue inhibitor of MMP (TIMP) in myometrial smooth muscle cells. Using primary cultures of human myometrial smooth muscle cells we found that these cells express MMP-1, MMP-3, TIMP-1 and TIMP-2 mRNA and protein, with significantly higher values of TIMP than MMP. We also found that TGF-beta1 (1 ng/ml) increased the expression of TIMP-1 mRNA, while it reduced the expression of MMP-1 and MMP-3 mRNA, compared with untreated controls. In addition, TGF-beta1 slightly increased the production of TIMP-1, but not TIMP-2. Production of MMP-1 and MMP-3 was reduced by treatment with TGF-beta1, compared with the untreated control. A major portion of MMP-1 released into the culture-conditioned media was in complex with TIMP-1, and the levels of this complex were reduced by treatment with TGF-beta1. In conclusion, the data indicate that myometrial smooth muscle cells express MMP and TIMP mRNA and protein, and their expression is differentially regulated by TGF-beta1. Such a differential regulation of MMP and TIMP by TGF-beta may influence the rate of extracellular matrix (ECM) turnover following tissue injury, induced during myomectomy and Caesarean section, or in leiomyomas during growth.     

A 90-kd surface antigen from a subpopulation of smooth muscle cells from human atherosclerotic lesions.

A monoclonal antibody, designated 10F3, that reacts with an antigen with a molecular weight of 90,000 daltons has been obtained after immunization of BALB/c mice with long-term cultured smooth muscle cells (SMC) originally isolated from fetal human aorta (fSMC). In adults the antigen is present on venous, arterial and capillary endothelial cells of heart, kidney, liver, spleen, intestine, skin, uterus, placenta, and arteries only, as shown by immunohistochemical investigation using the PAP technique. The antigen 10F3 is also present on the mesenchymal cells of human fetal tissues (7 and 18-week-old fetuses) and on SMC of 7-week-old fetal aorta, and a subpopulation of cells reacting with 10F3 antibody also has been found in atherosclerotic intima. Double staining using 10F3 antibody and muscle actin-specific monoclonal antibody HHF-35 showed that the antigen-positive cells are smooth muscle cells. In primary culture of adult SMC, antigen-positive cells were detected 2 days after seeding (about 90% positive in medial and intimal cultures). It is suggested that 10F3 is a mesenchymal antigen that, lost during differentiation by cells other than endothelium, but expressed again by the SMC involved in atherogenesis.     

Active macrophage-associated TGF-beta co-localizes with type I procollagen gene expression in atherosclerotic human pulmonary arteries.

Vascular remodeling in adult atherosclerotic pulmonary arteries is characterized by discrete areas of neointimal smooth muscle cell extracellular matrix gene expression in close proximity to non-foamy macrophages, suggesting regulation by local macrophage-associated factors. The purpose of these studies was to begin addressing the role of putative macrophage-associated factors such as transforming growth factor-beta (TGF-beta), by determining the spatial relationship between TGF-beta and neointimal matrix gene expression in human atherosclerotic pulmonary arteries. For example, the participation of TGF-beta in vascular remodeling could be inferred by its colocalization with non-foamy macrophages in areas of active matrix synthesis. In situ hybridization and immunohistochemistry demonstrated focal neointimal procollagen gene expression in close association with non-foamy but not foamy macrophages. Immunohistochemistry with isoform-specific anti-TGF-beta antibodies demonstrated all three isoforms of TGF-beta associated with non-foamy macrophages, but foamy macrophages were not immunoreactive. Neointimal and medial smooth muscle cells stained lightly. In contrast, intense TGF-beta immunoreactivity was also associated with medial smooth muscle cells in normal nonremodeling vessels. Immunohistochemistry with antibodies specific for latent TGF-beta was similar to immunohistochemistry for mature TGF-beta in both remodeling and nonremodeling vessels. Finally, using an antibody specific for active TGF-beta 1, immunoreactivity was only seen in non-foamy neointimal macrophages but not in foamy macrophages or medial smooth muscle cells from hypertensive or normal vessels. These observations suggest non-foamy macrophages may participate in modulating matrix gene expression in atherosclerotic remodeling via a TGF-beta-dependent mechanism.     

The effect of enzymatically degradable poly(ethylene glycol) hydrogels on smooth muscle cell phenotype

The formation of scar tissue due to dedifferentiation of smooth muscle cells (SMCs) is one of the major issues faced when engineering bladder tissue. Furthermore, cell sources for regenerating the SMC layer are also limiting. Here we explore if human mesenchymal stem cells (MCSs), cultured in enzymatically degradable poly(ethylene glycol) (PEG) hydrogel scaffolds can be differentiated into SMC-like cells. We explored the degree to which a less synthetic SMC phenotype can be achieved when primary human SMCs are cultured within these scaffolds, It was observed that when both MSCs and SMCs are cultured in the PEG hydrogel scaffolds, but not on traditional tissue culture plastic, they up-regulate markers associated with the less synthetic SMC phenotype, decreased expression of alpha(5) integrin and THY-1, and increased expression of alpha-smooth muscle actin (alphaSMA) and myosin. Furthermore, we show that MSCs and SMCs cultured in the PEG hydrogels are able to proliferate and express matrix metalloproteinases for up to 21d in culture, the duration of the study. This study addresses the importance of the cellular microenvironment on cell fate, and proposes synthetic instructive biomaterials as a means to direct cell differentiation and circumvent scar tissue formation during bladder reconstruction.     

Endothelial-mesenchymal interactions in vitro reveal molecular mechanisms of smooth muscle/pericyte differentiation

Cell-cell interactions are central to vascular development. We have developed an in vitro system in which endothelial cells (EC) are co-cultured with 10T1/2 cells as smooth muscle cell (SMC)/pericyte precursors. 10T1/2 cells, in contact with EC, differentiate to SMC in a process mediated, at least in part, by a transforming growth factor-beta (TGF-beta)-mediated event. Co-culture with EC or TGF-beta treatment induced expression of SM22alpha, with co-culture inducing a significantly greater response. To dissect the molecular mechanisms of SMC/pericyte differentiation, reporter constructs containing the promoter for SM22alpha, a SMC-specific gene, were stably transfected into 10T1/2 cells and response to EC-co-culture and TGFbeta were compared. Co-culture with EC or TGFbeta treatment stimulated activity of a 441-bp SM22-alpha promoter to about the same extent, whereas co-culture induced the activity of a 3.7-kb promoter to about twice that of TGBbeta. Neutralization of TGFbeta in EC-10T1/2 co-cultures partially reduced the 3.7-kb SM22alpha promoter activity in 10T1/2 cells. Previously unidentified CArG and TCE elements near the 5' end of the promoter are responsible for full promoter activity. EC-mesenchymal contact appears to be required for full promoter activity of the SM22alpha gene in 10T1/2 and requires upstream CArG and TCE elements. The 3.7-kb SM22alpha promoter can direct expression of lacZ in vivo to SMC of the large vessels and the smaller intersomitic vessels. We have identified the expression of SM22alpha in pericytes of the retinal microvasculature in developing and remodeling vessels.     

Smooth muscle homeostasis in human atherosclerotic plaques through interleukin 15 signalling

Interleukin (IL)-15 is a cytokine that has a broad tissue distribution and is important in maintaining homeostasis of cells and stability of tissues. When II-15 is also expressed by vascular smooth muscle cells (SMC), which are the dominant type of cells in most atherosclerotic plaques, it could be important in maintaining plaque tissue integrity and hence resistance of plaques towards development of clinically relevant complications such as plaque rupture and thrombosis. In this study, IL-15 and IL-15Rα in vitro expression by coronary artery SMC was investigated using RT -PCR and FACS analysis. Immunohistochemistry was used to study in situ expression of IL-15 and IL-15R by SMC of human carotid artery atherosclerotic plaques. Multiplex ligand-dependent probe amplification (MLPA) was used to investigate the mRNA expression of 40 pro- and anti inflammatory genes after stimulating coronary SMC with IL-15. We found that atherosclerotic SMC express both IL-15 and its receptor IL-15R, and TNF-γ and TNF-α enhance IL-15R expression in cultured SMC. MLPA studies on SMC revealed enhanced expression of PDGF beta mRNA after IL15 stimulation. In conclusion, our data suggest that IL-15 may contribute to atherosclerotic plaque integrity by stimulation of smooth muscle cells, probably in a PDGF dependent fashion.     

Role of reactive oxygen species in vascular remodeling associated with pulmonary hypertension

Several manifestations of neonatal pulmonary hypertension are associated with vascular remodeling, resulting in increased muscularity of the small pulmonary arteries. Abnormal structural development of the pulmonary vasculature has been implicated in persistent pulmonary hypertension of the newborn (PPHN). Increased plasma levels of the vasoconstrictor endothelin-1 (ET-1) have been demonstrated in patients with PPHN, which is likely to contribute to hypertension. In addition, several studies have identified a role for ET-1 in the proliferation of vascular smooth muscle cells (SMCs), suggesting that ET-1 may also be involved in the vascular remodeling characteristic of this disease. However, the mechanisms of ET-1-induced SMC proliferation are unclear and appear to differ between cells from different origins within the vasculature. In SMCs isolated from fetal pulmonary arterial cells, ET-1 stimulated proliferation via an induction of reactive species (ROS). Furthermore, other lines of evidence have demonstrated the involvement of ROS in ET-1-stimulated SMC growth, suggesting that ROS may be a common factor in the mechanisms involved. This review discusses the potential roles for ROS in the abnormal pulmonary vascular development characteristic of PPHN, and the treatment strategies arising from our increasing knowledge of the molecular mechanisms involved.     

Smad protein and TGF-beta signaling in vascular smooth muscle cells

Transforming growth factor-beta1 (TGF-beta1) plays a role in vascular remodeling by stimulating vascular smooth muscle cell (SMC) growth and matrix-protein synthesis at sites of vascular injury. Smad proteins have been shown to mediate intracellular signaling of this growth factor. We investigated the expression and phosphorylation of Smads in cultured rat aortic smooth muscle cells. In addition, we evaluated the effects of overexpression of Smad proteins on TGF-beta signal transduction by adenovirus-mediated gene transfer. In rat SMC, Smad1, Smad2, Smad3, Smad4 and Smad5 were detected by immunoprecipitation. Using antisera against phosphorylated Smad2, we showed that TGF-beta1-induced Smad2 phosphorylation in a concentration- and time-dependent manner. Using adenovirus-mediated transfection method, we demonstrated that overexpression of Smad2 or Smad4 was associated with an increased production of TGF-beta1-induced plasminogen activator inhibitor-1 (PAI-1). However, the most prominent expression of PAI-1 was observed upon cotransfection of both Smad2 and Smad4. Both the proliferative effect of TGF-beta1 under serum-free conditions and its anti-proliferative effect under serum-rich conditions were suppressed by the adenovirus-mediated overexpression of Smad7. These results indicated that Smads proteins were expressed in vascular SMC and that they mediated TGF-beta signaling in those cells.     

Differentiation of bone marrow mesenchymal stem cells into the smooth muscle lineage by blocking ERK/MAPK signaling pathway

Smooth muscle cells (SMCs) are major components of blood vessels and other hollow visceral organs required for tissue engineering of these organs. This study aims to evaluate whether adult bone marrow-derived mesenchymal stem cells (BMMSCs), multipotent cells, can be converted into SMCs. We examined the ERK/MAPK pathway as it exerts anti-myogenic signals in SMCs. Undifferentiated BMMSCs express most SMC marker genes, albeit mainly at low levels, except smooth muscle myosin heavy chain (SMMHC), the most definitive marker of differentiated SMC. The treatment of BMMSC with MEK inhibitor up-regulated the expression of alpha-smooth muscle actin (ASMA), h-caldesmon, and SMMHC in BMMSC in low serum condition. MEK inhibitor-treated BMMSC also contracted a collagen gel in response to endothelin. Interestingly, inhibition of MEK induced myocardin expression in BMMSC. In conclusion, BMMSCs treated MEK inhibitor gain a SMC-like phenotype with ligand-induced cell contractility to endothelin in vitro. This approach has obvious implications for cell therapeutics and tissue engineering of hollow visceral organs such as blood vessels.     

Dynamic expression of alpha 1 beta 1 and alpha 2 beta 1 integrin receptors by human vascular smooth muscle cells. Alpha 2 beta 1 integrin is required for chemotaxis across type I collagen-coated membranes.

Vascular smooth muscle cells (SMCs) in the media of normal arteries express alpha 1 beta 1 integrin with no detectable alpha 2 beta 1 as determined by immunocytochemistry. In contrast, immunoprecipitation of integrins expressed by human SMCs cultured from medial explants shows strong expression of alpha 2 beta 1 and no expression of alpha 1 beta 1. The apparent reciprocal expression of these two collagen and laminin receptors was confirmed by flow cytometric analysis of fluorescent labeled cells. Freshly isolated SMCs had detectable alpha 1, alpha 3, alpha 5, and alpha v subunits with low levels of detectable beta 3 and no detectable alpha 2. Cultured SMCs expressed alpha 2, alpha 3, alpha 5 and alpha v subunits with little or no alpha 1 or beta 3. Neither alpha 4 nor alpha 6 were detectable in freshly isolated or cultured cells. Expression of alpha 2 beta 1 receptors by cultured SMCs appears to be required for the migration of these cells on type I collagen. Migration of cultured SMCs across a type I collagen-coated membrane toward two different chemotactic stimuli, platelet-derived growth factor-BB (1 nmol/L) and insulin-like growth factor-(1 nmol/L), was Mg2+ dependent and inhibited by preincubation of cells with an affinity-purified polyclonal anti-alpha 2 beta 1 antibody or by monoclonal antibodies directed against the individual alpha 2 or beta 1 subunits. Attachment to type 1 collagen membranes was not affected by antibodies under conditions where migration was significantly impeded. The combined data show that SMC expression of alpha 1 beta 1 and alpha 2 beta 1 integrin receptors for collagen and laminin is dynamic and reciprocal and may be important with respect to SMC migration on type I collagen. These findings are potentially important in understanding the pathophysiology of vascular diseases, for example, atherosclerosis and restenosis following balloon angioplasty, where SMC migration is a contributing factor.     

Endothelin-2 is a macrophage chemoattractant: implications for macrophage distribution in tumors

Endothelins (ET-1, ET-2 and ET-3) are 21-amino acid vasoactive peptides that bind to G-protein-linked transmembrane receptors, ET-RA and ET-RB. As well as modulating vasoconstriction, endothelins regulate growth in several cell types and may also affect differentiation, inflammation and angiogenesis. Both macrophages and endothelins are found in areas of hypoxia in solid tumors and ET-2 expression may be modulated by hypoxia in some tumors. As the peptide structure of mature endothelins is similar to that of CXC chemokines, we asked if endothelins contribute to control of macrophage distribution in tumors. We found that ET-2 is a chemoattractant for macrophages and THP-1 monocytic cells, but not for freshly isolated monocytes. The chemotactic response to ET-2 shows a typical bell-shaped response curve. Experiments with endothelin receptor antagonists showed that migration to ET-2 is mediated via the ET-RB receptor. Moreover, monocytes do not express ET-RB. Chemotaxis towards ET-2 is via the MAPK pathway: p44 and p42 are phosphorylated when THP-1 cells are stimulated with ET-2, and the MAPKK inhibitor PD98059 stops chemotaxis. As with 'classical' chemokines, migration toET-2 is also inhibited by hypoxia and by pertussis toxin. As well as its chemotactic properties, ET-2 leads to activation of macrophages. In human breast tumors that express ET-2, endothelins and ET-RB expressing macrophages often co-localized. While shorter than 'classical' chemokines, ET-2 shares a similar peptide sequence with chemokines and may signal via a similar receptor and MAPK-mediated pathway. Furthermore, ET-2 expression by tumors may modulate the behavior of macrophages such that activated cells accumulate in areas of hypoxia.     

CCNs, fibulin-1C and S100A4 expression in leiomyoma and myometrium: inverse association with TGF-beta and regulation by TGF-beta in leiomyoma and myometrial smooth muscle cells

Connective tissue growth factor (CTGF; CCN2) is considered to serve as downstream midiator of TGF-beta action in tissue fibrosis. We tested this hypothesis in paired leiomyoma and myometrium by evaluating the expression of TGF-beta1/TGF-beta3 and CCN2, the other members of the CCN family, CCN3 and CCN4, as well as fibulin-1C and S100A4, calcium-binding proteins that interact with CCNs. The regulatory function of TGF-beta1 on the expression of these genes was further evaluated using leiomyoma (L) and myometrial (M) smooth muscle cells (SMC). Real-time PCR, Western blotting and immunohistochemistry revealed that leiomyomas and myometrium express CCNs, fibulin-1C and S100A4, whose levels of expression with the exception of fibulin-1C were lower in leiomyomas and inversely correlated with the expression of TGF-beta1 and TGF-beta3 (P<0.05). The expression of these genes was menstrual cycle-independent and GnRHa therapy increased the expression of CCN2 in leiomyomas, while inhibiting CCN3, CCN4 and S100A4 in myometrium (P<0.05). TGF-beta (2.5 ng/ml) in a time- and cell-dependent manner, and through MAPK and Smad pathways, differentially regulated the expression of these genes in LSMC and MSMC. We concluded that CCNs, fibulin-1C and S100A4 are expressed in leiomyomas/myometrium with relative expression levels inversely correlating with TGF-betas and influenced by GnRHa and TGF-beta regulatory actions. The results suggest that unlike other fibrotic disorders, CCN2 (CTGF), at least at tissue level, may not serve as a downstream mediator of TGF-beta action in leiomyomas.     

Macrophage colony-stimulating factor gene expression in vascular cells and in experimental and human atherosclerosis.

The infiltration of monocytes into the vascular wall and their transformation into lipid-laden foam cells characterizes early atherogenesis. Macrophages are also present in more advanced human atherosclerotic plaques and can produce many mediators that may contribute to lesion formation and progression. Macrophage colony-stimulating factor (MCSF) enhances the proliferation and differentiation of monocyte progenitors and is required for the survival and activation of mature monocytes and macrophages. The authors therefore examined the expression of the MCSF gene in cultured human vascular endothelial (EC) and smooth muscle cells (SMC) as well as in atheromatous lesions from rabbits and humans. Growth arrested EC and SMC contain a low level of MCSF mRNA. Bacterial lipopolysaccharide (LPS), recombinant human interleukin-1 alpha (IL-1 alpha) or tumor necrosis factor alpha (TNF alpha) induced MCSF mRNA accumulation in a concentration-dependent manner in both EC and SMC. These stimuli induced large increases in MCSF mRNA with peak induction between 4-8 hours after treatment. LPS, IL-1 alpha, and TNF alpha stimulated EC and SMC also showed increased fluorescent antibody staining for MCSF protein and released immunoreactive MCSF in a time-dependent manner. In contrast, phorbol 12-myristate 13-acetate (PMA) was a less potent inducer of MCSF gene expression and iron-oxidized low-density lipoproteins (ox-LDL) did not increase consistently MCSF mRNA or the synthesis and secretion of immunoreactive protein. Northern analysis of mRNA isolated from the atheromatous aorta of rabbits fed a 1% cholesterol diet for 10 weeks showed elevated MCSF mRNA compared with controls. Immunostaining of atheromatous arterial lesions of rabbits demonstrated MCSF protein in association with intimal SMC as well as macrophages. Furthermore, polymerase chain reaction (PCR) analysis of MCSF mRNA in human atheromata showed higher levels than found in nonatherosclerotic arteries and veins. Since the authors found no mRNA for the MCSF receptor, c-fms, in cultured EC or SMC macrophages are likely the primary target for MCSF within atheromatous vessels. The authors therefore investigated the effects of MCSF on monocyte functions related to foam cell development. Treatment of cultured human monocytes with recombinant human MCSF (10(3) U/ml, 72 hr) led to the accumulation of mRNA for the acetyl-LDL (scavenger) receptor and apolipoprotein E (apo E). These studies establish that vascular EC and SMC produce substantial MCSF in response to a variety of stimuli. The local production of MCSF during atherogenesis may contribute to macrophage survival and proliferation or activate specific macrophage functions such as expression of the scavenger receptor and secretion of apo E.     

Vascular smooth muscle cells for use in vascular tissue engineering obtained by endothelial-to-mesenchymal transdifferentiation (EnMT) on collagen matrices

The discovery of the endothelial progenitor cell (EPC) has led to an intensive research effort into progenitor cell-based tissue engineering of (small-diameter) blood vessels. Herein, EPC are differentiated to vascular endothelial cells and serve as the inner lining of bioartificial vessels. As yet, a reliable source of vascular smooth muscle progenitor cells has not been identified. Currently, smooth muscle cells (SMC) are obtained from vascular tissue biopsies and introduce new vascular pathologies to the patient. However, since SMC are mesenchymal cells, endothelial-to-mesenchymal transdifferentiation (EnMT) may be a novel source of SMC. Here we describe the differentiation of smooth muscle-like cells through EnMT. Human umbilical cord endothelial cells (HUVEC) were cultured either under conditions favoring endothelial cell growth or under conditions favoring mesenchymal differentiation (TGF-beta and PDGF-BB). Expression of smooth muscle protein 22alpha and alpha-smooth muscle actin was induced in HUVEC cultured in mesenchymal differentiation media, whereas hardly any expression of these markers was found on genuine HUVEC. Transdifferentiated endothelial cells lost the ability to prevent thrombin formation in an in vitro coagulation assay, had increased migratory capacity towards PDGF-BB and gained contractile behavior similar to genuine vascular smooth muscle cells. Furthermore, we showed that EnMT could be induced in three-dimensional (3D) collagen sponges. In conclusion, we show that HUVEC can efficiently transdifferentiate into smooth muscle-like cells through endothelial-to-mesenchymal transdifferentiation. Therefore, EnMT might be used in future progenitor cell-based vascular tissue engineering approaches to obtain vascular smooth muscle cells, and circumvent a number of limitations encountered in current vascular tissue engineering strategies.     

Hypoxic induction of cox-2 regulates proliferation of human pulmonary artery smooth muscle cells

Chronic hypoxia-induced pulmonary hypertension results partly from proliferation of smooth muscle cells in small peripheral pulmonary arteries. Therefore, we examined the effect of hypoxia on growth of pulmonary artery smooth muscle cells (PASMCs) from human distal pulmonary arteries. Initial studies identified that serum-induced proliferation of explant-derived PASMCs was inhibited under hypoxic conditions (3-4 kPa in medium). However, selection of hypoxia-stimulated cells was achieved by culturing cells at low density under conditions of prolonged hypoxia (1-2 wk). In hypoxia-inhibited and -stimulated cells, Western blotting revealed hypoxic induction of cyclooxygenase (COX)-2, which was dependent on the activation of p38(MAPK), but not COX-1, inducible nitric oxide synthase (iNOS), or hemoxygenase-1 (HO-1). Hypoxic induction of COX-2 was also observed in the media of pulmonary arteries in lung organ culture. Hypoxia induced a 4- to 5-fold increase (P < 0.001) in prostaglandin (PG)E(2), PGD(2), PGF(2alpha), and 6-keto-PGF(1alpha) release from PASMCs. Hypoxic inhibition of proliferation was attenuated by incubation with indomethacin (10 micro M), or the COX-2 antagonist, NS398 (10 micro M), but not by the COX-1 antagonist, valeryl salicylate (0.5 mM). In conclusion, we have isolated cells from human peripheral pulmonary arteries that are either inhibited or stimulated by culture under hypoxic conditions. In both cell types hypoxia modulates cell proliferation by induction of COX-2 and production of antiproliferative prostaglandins. Induction of COX-2 may contribute to the inhibition of hypoxia-induced pulmonary vascular remodeling.     

Inducible expression of vascular cell adhesion molecule-1 by vascular smooth muscle cells in vitro and within rabbit atheroma.

Vascular cell adhesion molecule-1 (VCAM-1), a mononuclear leukocyte adhesion molecule, is expressed in cultured vascular endothelial cells activated by cytokines and is induced in rabbit aortic endothelium in vivo within 1 week after initiation of an atherogenic diet. We now demonstrate that vascular smooth muscle cells can also express VCAM-1 in rabbit atherosclerotic lesions in vivo and in response to cytokines in vitro. Immunohistochemical staining of aortas from rabbits fed a 0.3% cholesterol-containing diet revealed that a portion of smooth muscle cells within intimal foam cell-rich lesions expressed VCAM-1. The intimal VCAM-1-expressing cells localized predominantly in regions above the internal elastic lamina. These VCAM-1-positive cells had the typical spindle shape of smooth muscle cells but had reduced alpha-actin expression in comparison to normal medial smooth muscle cells, and did not bear markers for endothelium, macrophages, and T cells. In culture, rabbit aortic smooth muscle cells expressed VCAM-1 mRNA and protein in a time- and concentration-dependent fashion when exposed to interferon-gamma or Gram-negative bacterial lipopolysaccharide. Cultured human vascular smooth muscle cells also expressed VCAM-1 mRNA and protein in response to lipopolysaccharide, interferon-gamma, and interleukin-4. The monokines interleukin-1 alpha and tumor necrosis factor-alpha did not induce VCAM-1 expression in either rabbit or human vascular smooth muscle cells. Inducible VCAM-1 expression by vascular smooth muscle cells in vivo during hypercholesterolemia and in vitro in response to certain cytokines suggests a broader range of VCAM-1 functions in vascular biology than heretofore appreciated.