Comparison of the effect of ATP on intracellular calcium ion dynamics between rat testicular and cerebral arteriole smooth muscle cells

Adenosine 5'-triphosphate (ATP), when released from neuronal and non-neuronal tissues, interacts with cell surface receptors produces a broad range of physiological responses. The goal of the present study was to examine the issue of whether vascular smooth muscle cells respond to ATP. To this end, the dynamics of the intracellular concentration of calcium ions ([Ca(2+)](i)) in smooth muscle cells in testicular and cerebral arterioles was examined by laser scanning confocal microscopy. ATP produced an increase in [Ca(2+)](i) in arteriole smooth muscle cells. While P1 purinoceptor agonists had no effect on this process, P2 purinoceptor agonists induced a [Ca(2+)](i) increase and a P2 purinoceptor antagonist, suramin, completely inhibited ATP-induced [Ca(2+)](i) dynamics in both arteriole smooth muscle cells.In testicular arterioles, Ca(2+) channel blockers and the removal of extracellular Ca(2+), but not thapsigargin pretreatment, abolished the ATP-induced [Ca(2+)](i) dynamics. In contrast, Ca(2+) channel blockers and the removal of extracellular Ca(2+) did not completely inhibit ATP-induced [Ca(2+)](i) dynamics in cerebral arterioles. Uridine 5'-triphosphate caused an increase in [Ca(2+)](i) only in cerebral arterioles and alpha,beta-methylene ATP caused an increase in [Ca(2+)](i) in both testicular and cerebral arterioles.We conclude that testicular arteriole smooth muscle cells respond to extracellular ATP via P2X purinoceptors and that cerebral arteriole smooth muscle cells respond via P2X and P2Y purinoceptors.     

Recruitment of smooth muscle cells and arterial vasomotion

Investigating the recruitment and synchronization of smooth muscle cells (SMCs) is the key to understanding the physical mechanisms leading to contraction and spontaneous diameter oscillations of arteries, called vasomotion. We improved a method that allows the correlation of calcium oscillations (flashing) of individual SMCs with mean calcium variations and arterial contraction using confocal microscopy. Endothelium-stripped rat mesenteric arteries were cut open, loaded with dual calcium fluorescence probes, and stimulated by increasing concentrations of the vasoconstrictors phenylephrine (PE) and KCl. We found that the number and synchronization of flashing cells depends on vasoconstrictor concentration. At low vasoconstrictor concentration, few cells flash asynchronously and no local contraction is detected. At medium concentration, recruitment of cells is complete and synchronous, leading to strip contraction after KCl stimulation and to vasomotion after PE stimulation. High concentration of PE leads to synchronous calcium oscillations and fully contracted vessels, whereas high concentration of KCl leads to a sustained nonoscillating increase of calcium and to fully contracted vessels. We conclude that the number of simultaneously recruited cells is an important factor in controlling rat mesenteric artery contraction and vasomotion.     

Stimulation of Leydig cell function by a polypeptide present in testicular interstitial fluid

Rat testicular interstitial fluid (IF) contains a polypeptide factor(s) which when added to Percoll-purified rat Leydig cells in vitro, enhances both basal and hCG-stimulated production of testosterone in a dose-dependent manner. To assess the possible physiological significance of this factor(s), its levels in testicular IF from individual rats were assessed following the suppression of intratesticular levels of testosterone by either (a) treatment with anti-LH, (b) induction of unilateral cryptorchidism, or (c) treatment with ethane-1,2-dimethane sulphonate (EDS). These treatments suppressed testosterone levels in IF by 80 to 99% and in each instance levels of the IF-factor were increased. This increase occurred as early as 5h after anti-LH treatment, but restoration of normal intratesticular levels of testosterone at 20-40 h after anti-LH treatment by testosterone injection failed to normalize levels of the IF-factor. In contrast, injection of LH into normal rats raised IF testosterone to supranormal levels and this was associated with a reduction in levels of the IF-factor, and similar effects of hCG were observed in rats pretreated with anti-LH. Therefore, whilst these studies suggest that the IF-factor(s) may be of physiological importance, its local regulation does not appear to involve a straightforward negative feedback control by testosterone.     

Uhrf2 is important for DNA damage response in vascular smooth muscle cells

Emerging evidence shows that Uhrf1 plays an important role in DNA damage response for maintaining genomic stability. Interestingly, Uhrf1 has a paralog Uhrf2 in mammals. Uhrf1 and Uhrf2 share similar domain architectures. However, the role of Uhrf2 in DNA damage response has not been studied yet. During the analysis of the expression level of Uhrf2 in different tissues, we found that Uhrf2 is highly expressed in aorta and aortic vascular smooth muscle cells. Thus, we studied the role of Uhrf2 in DNA damage response in aortic vascular smooth muscle cells. Using laser microirradiation, we found that like Uhrf1, Uhrf2 was recruited to the sites of DNA damage. We dissected the functional domains of Uhrf2 and found that the TTD, PHD and SRA domains are important for the relocation of Uhrf2 to the sites of DNA damage. Moreover, depletion of Uhrf2 suppressed DNA damage-induced H2AX phosphorylation and DNA damage repair. Taken together, our results demonstrate the function of Uhrf2 in DNA damage response.     

PDGF-AA activates AKT and ERK signaling for testicular interstitial Leydig cell growth via primary cilia

Testes control the development of male reproductive system. The testicular interstitial Leydig cells (Leydig cells) synthesize testosterone for promoting spermatogenesis and secondary sexual characteristics. Type A platelet-derived growth factor (PDGF-AA) is one of the most important growth factors in regulating Leydig cell growth and function. Knockout of PDGF-AA or its congenital receptor PDGFR-α leads to poor testicular development caused by reducing Leydig cell numbers, supporting PDGF-AA/PDGFR-α signaling regulates Leydig cell development. Primary cilium is a cellular antenna that functions as an integrative platform to transduce extracellular signaling for proper development and differentiation. Several receptors including PDGFR-α are observed on primary cilia for initiating signaling cascades in distinct cell types. Here we showed that PDGF-AA/PDGFR-α signaling promoted Leydig cells growth, migration, and invasion via primary cilia. Upon PDGF-AA treatment, AKT and ERK signaling were activated to regulate these cellular events. Interestingly, active AKT and ERK were detected around the base of primary cilia. Depletion of ciliary genes (IFT88 and CEP164) alleviated PDGF-AA-activated AKT and ERK, thus reducing Leydig cell growth, migration, and invasion. Thus, our study not only reveals the function of PDGF-AA/PDGFR-α signaling in maintaining testicular physiology but also uncovers the role of primary cilium and downstream signaling in regulating Leydig cell development.     

Fibrinogen is chemotactic for vascular smooth muscle cells

We studied the effect of fibrinogen on the migration of bovine aortic smooth muscle cells in culture, using a Neuro Probe 48-well micro chemotaxis chamber. Fibrinogen stimulated the migration of the cells dose-dependently at concentrations from 30 to 1000 micrograms/ml. A modified checkerboard analysis of the response demonstrated that the effect was largely chemotactic in nature. The present results suggest that fibrinogen may play an important role in the pathogenesis of arterial intimal thickening and atherosclerosis.     

Mechanical strain induces growth of vascular smooth muscle cells via autocrine action of PDGF

The effect of cyclic mechanical strain on growth of neonatal rat vascular smooth muscle (VSM) cells were examined. Cells were grown on silicone elastomer plates subjected to cyclic strain (60 cycle/min) by application of a vacuum under the plates. A 48 h exposure to mechanical strain increased the basal rate of thymidine incorporation by threefold and increased cell number by 40% compared with cells grown on stationary rubber plates. Strain also increased the rate of thymidine incorporation in response to alpha-thrombin (from 15- to 33-fold), but not to PDGF. As determined by thymidine autoradiography, strain alone induced a fourfold increase in labeled nuclei at the periphery of dishes, where strain is maximal, and a 2-3-fold increase at the center of dishes. Strain appeared to induce the production of an autocrine growth factor(s), since conditioned medium from cells subjected to strain induced a fourfold increase in DNA synthesis in control cells. Western blots of medium conditioned on the cells subjected to strain indicate that the cells secrete both AA and BB forms of PDGF in response to strain. Northern blots of total cell RNA from cells exposed to strain for 24 h show increased steady-state level of mRNA for PDGF- A. Lastly, polyclonal antibodies to the AA form of PDGF reduced by 75% the mitogenic effect of strain and polyclonal antibodies to AB-PDGF reduced mitogenicity by 50%. Antibodies to bFGF did not significantly reduce the strain-induced thymidine incorporation. Thus, the mechanism of strain-induced growth appears to involve the intermediary action of secreted PDGF.     

Changes in testicular hCG binding and Leydig cell function in rats throughout life

The age dependence of Leydig cell function was investigated in rats from prepuberty (15 days) to senescence (39 months). Serum LH, serum and testicular testosterone were measured by radioimmunoassay. The binding capacity and affinity of LH/hCG receptors were determined by a radioligand receptor assay (hCG/Leydig cells) using 125I-hCG labelled by the lactoperoxidase method. Separation of bound and free 125I and simultaneous concentrations of 125I-hCG was achieved by vacuum ultrafiltration. The biochemical integrity of 125I-hCG tracer was ascertained by various chromatographic procedures. The highest hCG-finding and highest serum LH levels were found during puberty. Serum and testicular testosterone concentrations, however, were maximal in early adulthood. From this period onwards to late senescence hCG-binding changed only slightly, while serum LH and testosterone levels decreased significantly towards late senescence. The study shows that, although hCG binding to the Leydig cell changes characteristically during development, it is minimally affected by aging and cannot therefore be responsible for the reduced androgen biosynthesis in senescence.     

Adenosine triphosphatases and related enzymes of the juxtaglomerular and arteriole smooth muscle cells in rat kidney

Synopsis Alkaline nucleoside diphosphatase, myosin ATPase and possibly an isozyme of the second, have been demonstrated histochemically at pH 9.4 in the arteriole smooth muscle cells and juxtaglomerular cells of the normal rat kidney.False negative results and non-specific reactions for calcium-activated ATPase have been prevented, at both the optical and the electron microscopical level, by adding cysteine to the incubating medium.Neither juxtaglomerular cells nor arteriole smooth muscle cells exhibited any histochemically demonstrable magnesium-activated ATPase, alkaline phosphatase or 5-nucleosidase.     

Characterization of Nestin-positive stem Leydig cells as a potential source for the treatment of testicular Leydig cell dysfunction

The ability to identify and isolate lineage-specific stem cells from adult tissues could facilitate cell replacement therapy. Leydig cells (LCs) are the primary source of androgen in the mammalian testis, and the prospective identification of stem Leydig cells (SLCs) may offer new opportunities for treating testosterone deficiency. Here, in a transgenic mouse model expressing GFP driven by the Nestin (Nes) promoter, we observed Nes-GFP⁺ cells located in the testicular interstitial compartment where SLCs normally reside. We showed that these Nes-GFP⁺ cells expressed LIFR and PDGFR-α, but not LC lineage markers. We further observed that these cells were capable of clonogenic self-renewal and extensive proliferation in vitro and could differentiate into neural or mesenchymal cell lineages, as well as LCs, with the ability to produce testosterone, under defined conditions. Moreover, when transplanted into the testes of LC-disrupted or aging models, the Nes-GFP⁺ cells colonized the interstitium and partially increased testosterone production, and then accelerated meiotic and post-meiotic germ cell recovery. In addition, we further demonstrated that CD51 might be a putative cell surface marker for SLCs, similar with Nestin. Taken together, these results suggest that Nes-GFP⁺ cells from the testis have the characteristics of SLCs, and our study would shed new light on developing stem cell replacement therapy for testosterone deficiency.     

Microtubule dynamics regulate cyclic stretch-induced cell alignment in human airway smooth muscle cells

Microtubules are structural components of the cytoskeleton that determine cell shape, polarity, and motility in cooperation with the actin filaments. In order to determine the role of microtubules in cell alignment, human airway smooth muscle cells were exposed to cyclic uniaxial stretch. Human airway smooth muscle cells, cultured on type I collagen-coated elastic silicone membranes, were stretched uniaxially (20% in strain, 30 cycles/min) for 2 h. The population of airway smooth muscle cells which were originally oriented randomly aligned near perpendicular to the stretch axis in a time-dependent manner. However, when the cells treated with microtubule disruptors, nocodazole and colchicine, were subjected to the same cyclic uniaxial stretch, the cells failed to align. Lack of alignment was also observed for airway smooth muscle cells treated with a microtubule stabilizer, paclitaxel. To understand the intracellular mechanisms involved, we developed a computational model in which microtubule polymerization and attachment to focal adhesions were regulated by the preexisting tensile stress, pre-stress, on actin stress fibers. We demonstrate that microtubules play a central role in cell re-orientation when cells experience cyclic uniaxial stretching. Our findings further suggest that cell alignment and cytoskeletal reorganization in response to cyclic stretch results from the ability of the microtubule-stress fiber assembly to maintain a homeostatic strain on the stress fiber at focal adhesions. The mechanism of stretch-induced alignment we uncovered is likely involved in various airway functions as well as in the pathophysiology of airway remodeling in asthma.     

Implications of autophagy for vascular smooth muscle cell function and plasticity

Vascular smooth muscle cells (VSMCs) are fundamental in regulating blood pressure and distributing oxygen and nutrients to peripheral tissues. They also possess remarkable plasticity, with the capacity to switch to synthetic, macrophage-like, or osteochondrogenic phenotypes when cued by external stimuli. In arterial diseases such as atherosclerosis and restenosis, this plasticity seems to be critical and, depending on the disease context, can be deleterious or beneficial. Therefore, understanding the mechanisms regulating VSMC phenotype and survival is essential for developing new therapies for vascular disease as well as understanding how secondary complications due to surgical interventions develop. In this regard, the cellular process of autophagy is increasingly being recognized as a major player in vascular biology and a critical determinant of VSMC phenotype and survival. Although autophagy was identified in lesional VSMCs in the 1960s, our understanding of the implications of autophagy in arterial diseases and the stimuli promoting its activation in VSMCs is only now being elucidated. In this review, we highlight the evidence for autophagy occurring in VSMCs in vivo, elaborate on the stimuli and processes regulating autophagy, and discuss the current understanding of the role of autophagy in vascular disease.     

Endothelin-induced contraction of bronchiole and pulmonary arteriole smooth muscle cells is regulated by intracellular Ca2+ oscillations and Ca2+ sensitization

Endothelin-1 (ET) induces increases in intracellular Ca(2+) concentration ([Ca(2+)](i)), Ca(2+) sensitization, and contraction of both bronchiole and pulmonary arteriole smooth muscle cells (SMCs) and may play an important role in the pathophysiology of asthma and pulmonary hypertension. However, because it remains unclear how changes in [Ca(2+)](i) and the Ca(2+) sensitivity regulate SMC contraction, we have studied mouse lung slices with phase-contrast and confocal microscopy to correlate the ET-induced contraction with the changes in [Ca(2+)](i) and Ca(2+) sensitivity of bronchiole and arteriole SMCs. In comparison with acetylcholine (ACh) or serotonin (5-HT), ET induced a stronger and long-lasting contraction of both bronchioles and arterioles. This ET-induced contraction was associated with prominent asynchronous Ca(2+) oscillations that were propagated as Ca(2+) waves along the SMCs. These Ca(2+) oscillations were mediated by cyclic intracellular Ca(2+) release and required external Ca(2+) for their maintenance. Importantly, as the frequency of the Ca(2+) oscillations increased, the extent of contraction increased. ET-induced contraction was also associated with an increase in Ca(2+) sensitivity. In "model" slices in which the [Ca(2+)](i) was constantly maintained at an elevated level by pretreatment of slices with caffeine and ryanodine, the addition of ET increased bronchiole and arteriole contraction. These results indicate that ET-induced contraction of bronchiole and arteriole SMCs is regulated by the frequency of Ca(2+) oscillations and by increasing the sensitivity of the contractile machinery to Ca(2+).     

NF-kappaB is required for TNF-alpha-directed smooth muscle cell migration.

Migration of vascular smooth muscle cells (VSMC) is a crucial event in the formation of vascular stenotic lesions. Tumor necrosis factor-alpha (TNF-alpha) is elaborated by VSMC in atherosclerosis and following angioplasty. We investigated the role of nuclear factor-kappaB (NF-kappaB) in human VSMC migration induced by TNF-alpha. Adenoviral expression of a mutant form of the inhibitor of NF-kappaB, IkappaB-alphaM, suppressed TNF-alpha-triggered degradation of cellular IkappaB-alpha, inhibited activation of NF-kappaB, and attenuated TNF-alpha-induced migration. Further, IkappaB-alphaM suppressed TNF-alpha-stimulated release of interleukin-6 and -8 (IL-6 and IL-8). Neutralization of IL-6 and IL-8 with appropriate antibodies reduced TNF-alpha-induced VSMC migration. Addition of recombinant IL-6 and IL-8 stimulated migration. Collectively, our data provide initial evidence that TNF-alpha-mediated VSMC migration requires NF-kappaB activation and is associated with induction of IL-6 and IL-8 which act in an autocrine manner.     

LKB1 in endothelial cells is required for angiogenesis and TGFbeta-mediated vascular smooth muscle cell recruitment

Inactivation of the tumor suppressor kinase Lkb1 in mice leads to vascular defects and midgestational lethality at embryonic day 9-11 (E9-E11). Here, we have used conditional targeting to investigate the defects underlying the Lkb1(-/-) phenotype. Endothelium-restricted deletion of Lkb1 led to embryonic death at E12.5 with a loss of vascular smooth muscle cells (vSMCs) and vascular disruption. Transforming growth factor beta (TGFbeta) pathway activity was reduced in Lkb1-deficient endothelial cells (ECs), and TGFbeta signaling from Lkb1(-/-) ECs to adjacent mesenchyme was defective, noted as reduced SMAD2 phosphorylation. The addition of TGFbeta to mutant yolk sac explants rescued the loss of vSMCs, as evidenced by smooth muscle alpha actin (SMA) expression. These results reveal an essential function for endothelial Lkb1 in TGFbeta-mediated vSMC recruitment during angiogenesis.     

Function of scavenger receptor class A type I/II is not important for smooth muscle foam cell formation

Macrophages (MPhi) and smooth muscle cells (SMC) are transformed into foam cells by massive accumulation of modified lipoproteins during atherogenesis. It is known that class AI/II scavenger receptors participate in the foam cell formation of MPhi. The mechanism of lipid accumulation in SMC is however unknown. Therefore, we investigated if class AI/II scavenger receptors mediate the uptake of modified lipoproteins in SMC. Additionally, we examined the influence of MPhi and proinflammatory cytokines in this process. Our flow cytometric experiments revealed significant uptake of DiI-AcLDL in SMC. This uptake was markedly enhanced by IL-1alpha and TNF-alpha, whereas cocultured MPhi decreased the uptake of DiI-AcLDL in SMC. Competition and blocking experiments were performed to enlighten the role of class AI/II scavenger receptors. The competition experiments showed that surplus NatLDL, a ligand not known to interact with class AI/II scavenger receptors, caused a drastically decreased uptake of DiI-AcLDL in SMC. Additionally, blocking of class AI/II scavenger receptors with antibody 2F8 did not influence the uptake of DiI-AcLDL in SMC. Furthermore, fluorescence microscopic double staining of human coronary arteries with early, intermediate and advanced atherosclerotic lesions showed no colocalization of class AI scavenger receptors with SMC. These results indicate that class AI/II scavenger receptors play only a minor role in the uptake of modified lipoproteins in SMC. We suggest that SMC foam cell formation is mainly mediated by other receptors than class AI/II scavenger receptors.     

Fgf10 expression identifies parabronchial smooth muscle cell progenitors and is required for their entry into the smooth muscle cell lineage

Lineage formation in the lung mesenchyme is poorly understood. Using a transgenic mouse line expressing LacZ under the control of Fgf10 regulatory sequences, we show that the pool of Fgf10-positive cells in the distal lung mesenchyme contains progenitors of the parabronchial smooth muscle cells. Fgf10 gene expression is slightly repressed in this transgenic line. This allowed us to create a hypomorphic Fgf10 phenotype by expressing the LacZ transgene in a heterozygous Fgf10 background. Hypomorphic Fgf10 mutant lungs display a decrease in beta-galactosidase-positive cells around the bronchial epithelium associated with an accumulation of beta-galactosidase-expressing cells in the distal mesenchyme. This correlates with a marked reduction of alpha smooth muscle actin expression, thereby demonstrating that FGF10 is mostly required for the entry of mesenchymal cells into the parabronchial smooth muscle cell lineage. The failure of exogenous FGF10 to phosphorylate its known downstream targets ERK and AKT in lung mesenchymal cultures strongly suggests that FGF10 acts indirectly on the progenitor population via an epithelial intermediate. We provide support for a role of epithelial BMP4 in mediating the formation of parabronchial smooth muscle cells.