Beneficial effects of platelet-derived growth factor on hemorrhagic shock in rats and the underlying mechanisms.

Studies have shown that local application of platelet-derived growth factor (PDGF) can be used for the treatment of acute and chronic wounds. We investigated if systemic application of PDGF has a protective effect on acute hemorrhagic shock in rats in the present study. Using hemorrhagic shock rats and isolated superior mesenteric arteries, the effects of PDGF-BB on hemodynamics, animal survival, and vascular reactivity as well as the roles of the gap junction proteins connexin (Cx)40 and Cx43, PKC, and Rho kinase were observed. PDGF-BB (1­15 µg/kg iv) significantly improved the hemodynamics and blood perfusion to vital organs (liver and kidney) as well as vascular reactivity and improved the animal survival in hemorrhagic shock rats. PDGF recovering shock-induced vascular hyporeactivity depended on the integrity of the endothelium and myoendothelial gap junction. Cx43 antisense oligodeoxynucleotide abolished these improving effects of PDGF, whereas Cx40 oligodeoxynucleotide did not. Further study indicated that PDGF increased the activity of Rho kinase and PKC as well as vascular Ca2+ sensitivity, whereas it did not interfere with the intracellular Ca2+ concentration in hypoxia-treated vascular smooth muscle cells. In conclusion, systemic application of PDGF-BB may exert beneficial effects on hemorrhagic shock, which are closely related to the improvement of vascular reactivity and hemodynamics. The improvement of PDGF-BB in vascular reactivity is vascular endothelium and myoendothelial gap junction dependent. Cx43, Rho kinase, and PKC play very important role in this process. These findings suggest that PDGF may be a potential measure to treat acute clinical critical diseases such as severe trauma, shock, and sepsis.

Increased arterial smooth muscle Ca2+ signaling, vasoconstriction, and myogenic reactivity in Milan hypertensive rats.

The Milan hypertensive strain (MHS) rats are a genetic model of hypertension with adducin gene polymorphisms linked to enhanced renal tubular Na(+) reabsorption. Recently we demonstrated that Ca(2+) signaling is augmented in freshly isolated mesenteric artery myocytes from MHS rats. This is associated with greatly enhanced expression of Na(+)/Ca(2+) exchanger-1 (NCX1), C-type transient receptor potential (TRPC6) protein, and sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA2) compared with arteries from Milan normotensive strain (MNS) rats. Here, we test the hypothesis that the enhanced Ca(2+) signaling in MHS arterial smooth muscle is directly reflected in augmented vasoconstriction [myogenic and phenylephrine (PE)-evoked responses] in isolated mesenteric small arteries. Systolic blood pressure was higher in MHS (145 ± 1 mmHg) than in MNS (112 ± 1 mmHg; P < 0.001; n = 16 each) rats. Pressurized mesenteric resistance arteries from MHS rats had significantly augmented myogenic tone and reactivity and enhanced constriction to low-dose (1-100 nM) PE. Isolated MHS arterial myocytes exhibited approximately twofold increased peak Ca(2+) signals in response to 5 µM PE or ATP in the absence and presence of extracellular Ca(2+). These augmented responses are consistent with increased vasoconstrictor-evoked sarcoplasmic reticulum (SR) Ca(2+) release and increased Ca(2+) entry, respectively. The increased SR Ca(2+) release correlates with a doubling of inositol 1,4,5-trisphosphate receptor type 1 and tripling of SERCA2 expression. Pressurized MHS arteries also exhibited a ∼70% increase in 100 nM ouabain-induced vasoconstriction compared with MNS arteries. These functional alterations reveal that, in a genetic model of hypertension linked to renal dysfunction, multiple mechanisms within the arterial myocytes contribute to enhanced Ca(2+) signaling and myogenic and vasoconstrictor-induced arterial constriction. MHS rats have elevated plasma levels of endogenous ouabain, which may initiate the protein upregulation and enhanced Ca(2+) signaling. These molecular and functional changes provide a mechanism for the increased peripheral vascular resistance (whole body autoregulation) that underlies the sustained hypertension.

Enhancement of receptor-operated cation current and TRPC6 expression in arterial smooth muscle cells of deoxycorticosterone acetate-salt hypertensive rats.

OBJECTIVES: In deoxycorticosterone acetate (DOCA)-salt hypertensive rats, altered reactivity of blood vessels to vasoactive agonists is frequently associated with an elevation in blood pressure. Canonical transient receptor potential (TRPC) channels are believed to encode receptor-operated cation channels (ROC), the activation of which is involved in smooth muscle depolarization and vasoconstriction. The aims of the present study were to investigate whether the ROC current is increased in DOCA-hypertensive rats and determine whether aldosterone directly enhances the expression of TRPC. METHODS: The nystatin-perforated patch-clamp technique was used for the recording of receptor-stimulated ion currents in mesenteric arterial smooth muscle cells, which were enzymatically dispersed from sham-operated and DOCA-salt hypertensive rats. Expressions of TRPCs were evaluated by reverse transcriptase-polymerase chain reaction (RT-PCR) and by Western blot analysis. RESULTS: Receptor-stimulated currents activated by 5-hydroxytryptamine (serotonin) and norepinephrine were increased significantly in the mesenteric arterial smooth muscle cells of DOCA-salt hypertensive rats compared to sham-operated rats. Ion-substitution experiments revealed that the enhanced currents were cation currents (ROC currents). Enhanced expression of TRPC6 in mesenteric arteries from DOCA-salt hypertensive rats was demonstrated by real-time RT-PCR. Up-regulation of TRPC6 by aldosterone treatment in vitro was also observed in A7r5 cells by RT-PCR and in western blots. CONCLUSION: These results suggest that aldosterone enhances TRPC6 expression and ROC currents in vascular smooth muscle cells, and that this may in turn contribute to altered vascular reactivity and to hypertension.

Poly (I:C) impairs NO donor-induced relaxation by overexposure to NO via the NF-kappa B/iNOS pathway in rat superior mesenteric arteries.

Recent studies have suggested a link between vascular dysfunction and innate immune activation including toll-like receptors (TLRs), but the detailed mechanism remains unclear. Here we investigated whether poly (I:C) [a synthetic double-strand RNA recognized by TLR3, melanoma differentiation-associated gene 5 (MDA5), and retinoic acid-inducible gene I (RIG-I)] affected nitric oxide (NO)/cGMP-related vascular relaxation, one of the major cascades of relaxation, in rat superior mesenteric arteries. Using organ-cultured arteries, we found that poly (I:C) (30µg/mL for approximately 1 day) markedly reduced sodium nitroprusside (SNP)-induced relaxation (vs. vehicle); this was prevented by co-treatment with a TLR3 inhibitor. Relaxation induced by 8-Br cGMP (a phosphodiesterase (PDE)-resistant cGMP analogue) and the expression of proteins related to NO/cGMP signaling did not differ between vehicle- and poly (I:C)-treated groups. When PDEs were inhibited by IBMX (a nonselective PDE inhibitor), the SNP-induced relaxation was still greatly reduced in poly (I:C)-treated arteries (vs. vehicle). Poly (I:C) reduced SNP-stimulated cGMP production, but increased NO production and iNOS expression (vs. vehicle). The impairment of SNP-induced relaxation by poly (I:C) was prevented by co-treatment with either iNOS or a nuclear factor-kappa B (NF-κB) inhibitor. This effect induced by poly (I:C) appeared to be independent of oxidative stress. The SNP-induced relaxation was reduced in freshly isolated arteries by pre-incubation with SNP in a concentration-dependent manner. Poly (I:C) did not alter protein levels of TLR3, TRIF/TICAM-1, or phospho-IRF3/IRF3, whereas RIG-I and MDA5 were significantly upregulated (vs. vehicle). These results suggest that poly (I:C) impairs NO donor-induced relaxation in rat superior mesenteric arteries via overexposure to NO produced by the NF-κB/iNOS pathway.

Membrane stretch directly activates large conductance Ca(2+)-activated K+ channels in mesenteric artery smooth muscle cells.

Large-conductance, Ca(2+)-activated K+ channels were identified in single smooth muscle cells freshly isolated from rabbit superior mesenteric artery. They typically showed a reversal potential close to 0 mV in excised, inside-out patches in symmetric 130 mmol/L [K+] with a unitary conductance of 260 pS, and increased activity at more positive potentials and/or when [Ca2+] was raised at the cytosolic surface of the membrane. Both in cell-attached and in excised, inside-out configurations, stretching the membrane patch by applying suction to the back of the patch pipette increased the activity of these channels without changing either the unitary conductance or the voltage sensitivity of the channel. Stretch activation was repeatedly seen in inside-out patches when both surfaces were bathed with a 0 Ca2+ solution containing 2 or 5 mmol/L EGTA to chelate trace amounts of Ca2+, making it highly improbable that stretch activation could be secondary to a stretch-induced flux of Ca2+. Consequently, stretch activation of large-conductance, Ca(2+)-activated K+ channels in mesenteric artery smooth muscle cells seems to be due to a direct effect of stretch on the channel itself or on some closely associated, membrane-bound entity.

Structure-activity relationships for relaxation of smooth muscle by VIP.

Utilizing VIP and five VIP analogues, concentration-response curves for relaxation of rat mesenteric artery and rat gastric longitudinal muscle were determined for comparison with our previously published radioligand binding data on rat smooth muscle and other tissues. The biological potency of the VIP analogues in the present study compared more closely with their potency for VIP receptor binding in smooth muscle tissue (arteries) vs. other tissues (pituitary, brain, liver).

Analysis of the time course for organ culture-induced endothelin ET B receptor upregulation in rat mesenteric arteries.

In contrast to the constitutively expressed endothelin ET(A) receptor, the distribution of endothelin ET(B) receptors is more variable. The aim of the present study was to investigate the kinetics of organ culture-induced upregulation of contractile endothelin ET(B) receptors in rat mesenteric arteries at both mRNA and functional levels. Assessment of mRNA expression revealed low levels of endothelin ET(B) receptor mRNA relative to endothelin ET(A) receptor mRNA after 3 h of culture, which gradually increased to reach a plateau level after 24 h. Correspondingly, vessels cultured for 3 h showed a negligible contractile response the selective endothelin ET(B) receptor agonist sarafotoxin 6c. Subsequently, the contractile response to sarafotoxin 6c was successively increased during organ culture until 24 h and, thereafter, a further increase in potency was seen after 48 h. These results demonstrate a rapid induction of transcription within less than 7 h followed by an increase in the response to receptor stimulation.

Role of gap junctions in endothelium-derived hyperpolarizing factor responses and mechanisms of K(+)-relaxation.

We have examined the effects of ouabain (1 mM), the gap junction inhibitors, 18 alpha-glycyrrhetinic acid (100 microM), N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2, 4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide hydrochloride (SR141716A; 10 microM) and palmitoleic acid (50 microM), and clotrimazole (10 microM) against endothelium-derived hyperpolarizing factor (EDHF)-mediated and K(+)-induced vasorelaxations in the rat mesentery. In the presence of indomethacin (10 microM) and 300-microM N(G)nitro-L-arginine methyl ester (L-NAME), carbachol caused EDHF-mediated relaxations (R(max)=85.3+/-4.0%). In the presence of ouabain, these responses were substantially reduced (R(max)=11.0+/-2.3%). 18 alpha-glycyrrhetinic acid, SR141716A, palmitoleic acid and clotrimazole also significantly inhibited these EDHF-mediated responses. K(+) caused vasorelaxation of preparations perfused with K(+)-free buffer (R(max)=73.7+/-2.4%), which were reduced by 10-microM indomethacin (R(max)=56.4+/-6.2%). K(+) vasorelaxation was essentially abolished by endothelial denudation. Both ouabain and 18 alpha-glycyrrhetinic acid opposed K(+) relaxations, however, neither SR141716A, clotrimazole nor palmitoleic acid had any effect. Direct cell-cell coupling via gap junctions was attenuated by ouabain, clotrimazole and palmitoleic acid. We conclude that: (i) that gap junctional communication plays a major role in EDHF-mediated relaxations, (ii) that K(+)-vasorelaxation is endothelium-dependent (thus, K(+) is unlikely to represent an EDHF), and (iii) that the inhibitory actions of ouabain and clotrimazole on gap junctions might contribute towards their effects against EDHF.

Histone deacetylase 4 controls neointimal hyperplasia via stimulating proliferation and migration of vascular smooth muscle cells.

Histone deacetylases (HDACs) are transcriptional coregulators. Recently, we demonstrated that HDAC4, one of class IIa family members, promotes reactive oxygen species-dependent vascular smooth muscle inflammation and mediates development of hypertension in spontaneously hypertensive rats. Pathogenesis of hypertension is, in part, modulated by vascular structural remodeling via proliferation and migration of vascular smooth muscle cells (SMCs). Thus, we examined whether HDAC4 controls SMC proliferation and migration. In rat mesenteric arterial SMCs, small interfering RNA against HDAC4 inhibited platelet-derived growth factor (PDGF)-BB-induced SMC proliferation as determined by a cell counting and bromodeoxyuridine incorporation assay as well as migration as determined by Boyden chamber assay. Expression and activity of HDAC4 were increased by PDGF-BB. HDAC4 small interfering RNA inhibited phosphorylation of p38 mitogen-activated protein kinase and heat shock protein 27 and expression of cyclin D1 as measured by Western blotting. HDAC4 small interfering RNA also inhibited PDGF-BB-induced reactive oxygen species production as measured fluorometrically using 2', 7'-dichlorofluorescein diacetate and nicotinamide adenine dinucleotide phosphate oxidase activity as measured by lucigenin assay. A Ca(2+)/calmodulin-dependent protein kinase II inhibitor, KN93, inhibited PDGF-BB-induced SMC proliferation and migration as well as phosphorylation of HDAC4. In vivo, a class IIa HDACs inhibitor, MC1568 prevented neointimal hyperplasia in mice carotid ligation model. MC1568 also prevented increased activation of HDAC4 in the neointimal lesions. The present results for the first time demonstrate that HDAC4 controls PDGF-BB-induced SMC proliferation and migration through activation of p38 mitogen-activated protein kinase/heat shock protein 27 signals via reactive oxygen species generation in a Ca(2+)/calmodulin-dependent protein kinase-dependent manner, which may lead to the neointimal hyperplasia in vivo.

Persufflation improves pancreas preservation when compared with the two-layer method.

Islet transplantation is emerging as a promising treatment for patients with type 1 diabetes. It is important to maximize viable islet yield for each organ due to scarcity of suitable human donor pancreata, high cost, and the large dose of islets required for insulin independence. However, organ transport for 8 hours using the two-layer method (TLM) frequently results in low islet yields. Since efficient oxygenation of the core of larger organs (eg, pig, human) in TLM has recently come under question, we investigated oxygen persufflation as an alternative way to supply the pancreas with oxygen during preservation. Porcine pancreata were procured from donors after cardiac death and preserved by either TLM or persufflation for 24 hours and subsequently fixed. Biopsies collected from several regions of the pancreas were sectioned, stained with hematoxylin and eosin, and evaluated by a histologist. Persufflated tissues exhibited distended capillaries and significantly less autolysis/cell death relative to regions not exposed to persufflation or to tissues preserved with TLM. The histology presented here suggests that after 24 hours of preservation, persufflation dramatically improves tissue health when compared with TLM. These results indicate the potential for persufflation to improve viable islet yields and extend the duration of preservation, allowing more donor organs to be utilized.

Unrepeatable extracellular Ca2+-dependent contractile effects of cyclopiazonic acid in rat vascular smooth muscle.

Cyclopiazonic acid (CPA), a specific reversible inhibitor of Ca(2+)-pumps in sarcoplasmic reticulum, causes a slowly developing and subsequently diminishing characteristic contraction in endothelium-denuded rat vascular smooth muscle. We recently found that CPA-induced contractions were not completely repeatable in endothelium-denuded rat aorta and superior mesenteric artery. 10 microM CPA-induced contractions expressed as a percentage of 80 mM KCl-induced contraction were significantly decreased from 51.4+/-5.7% to 11.8+/-2.6% (P<0.0001) upon the second application in endothelium-denuded rat aorta, and this was not due to any irreversible cytotoxic effects of CPA. The decrease of CPA-induced contractile responses upon the second application was dependent on both types of blood vessels and doses of CPA upon the first application. CPA upon the second application in Ca(2+)-containing solutions did induce its characteristic contractions in the rings pretreated with Ca(2+)-free solutions or Ca(2+) entry blockers before and during its first application, suggesting that capacitative mode of Ca(2+) influx during the application of CPA might be responsible for the diminishment of contractions upon the second application. These data suggest that CPA by inducing a transient rise in cytosolic Ca(2+) level might cause a long-lasting upregulation of Ca(2+) extrusion across the plasma membrane in vascular smooth muscle cells and thus accelerate Ca(2+) efflux over a prolonged period, leading to unrepeatable contractile effects of CPA. Such long-lasting upregulation of Ca(2+) extrusion may contribute to the regulation of excitability of vascular smooth muscle cells and protect the cells against excitotoxic injury.

Preparation of primary cultured mesenteric artery smooth muscle cells for fluorescent imaging and physiological studies.

In this protocol, we describe a method for isolation and culture of smooth muscle cells derived from the adult rat (or mouse) superior mesenteric artery. Arterial myocytes are obtained by enzymatic dissociation and established in primary culture. The cultured cells retain expression of smooth muscle-specific alpha-actin and physiological responses to agonists. Cultured arterial myocytes (prepared from wild-type or transgenic animals) provide a useful model for studying the regulation of a wide range of vascular smooth muscle responses at the cellular and subcellular levels. Plasmids, RNA interference and antisense oligodeoxynucleotides can be readily introduced into the cells to alter protein expression. Fluorescent dyes can also be introduced to visualize a variety of activities, some of which may be specific to vascular smooth muscle cells. This protocol requires about 3 h on each of 2 consecutive days to complete.

Three-dimensional characterization of dense bodies in contracted and relaxed mesenteric artery smooth muscle cells.

We have previously shown that dense bodies are not the static planar simple ovoidal structures they appear to be in thin sections. In this report, we present three-dimensional reconstructions from consecutive serial thin sections through shortened and non-shortened large mesenteric artery cells. Profiles of the cell surface, membrane dense bodies, and cytoplasmic dense bodies were reconstructed from consecutive thin sections and the distribution, size, shape, and spatial relationships among these components was examined. Within the cell, membrane dense bodies are numerous and occupy approximately 10% of the cell volume. Membrane dense bodies can attach to the cell surface laterally, obliquely or normally. An individual membrane dense body can be continuous over more than 2 microns of cell depth and can change shape throughout its depth. On cell shortening, many membrane dense bodies assume a crenated shape. Compared to membrane dense bodies, cytoplasmic dense bodies are smaller in all dimensions and occupy about 2% of the cell volume. In shortened cells, cytoplasmic dense bodies appear to cluster into groups. This redistribution of cytoplasmic dense bodies may be related to the reorganization of contractile units when the cell shortens.

Tumor necrosis factor expression in arterial walls of diabetic rats.

Immunohistochemistry was used to detect tumor necrosis factor (TNF-alpha) expression in arterial wall of diabetic rats. It was found that endothelial cells were swollen and markedly proliferative in these vessels and accordingly TNF-alpha showed strong positive immunohistochemical reaction in endothelial cells or extracellular intimal matrix of such vessels, which might be caused by the expression and release of TNF-alpha from monocytes and arterial wall cells stimulated by AGEs. These findings suggested that increased TNF-alpha expression might be associated with vascular damage and remodeling in diabetes.

Membrane ion channels as physiological targets for local Ca2+ signalling.

Ionized calcium plays a central role as a second messenger in a number of physiologically important processes determining smooth muscle function. To regulate a wide range of cellular activities the mechanisms of subcellular calcium signalling should be very diverse. Recent progress in development of visible light-excitable fluorescent dyes with high affinity for Ca2+ (such as oregon green 488 BAPTA indicators, fluo-3 and fura red) and confocal laser scanning microscopy provides an opportunity for direct visualization of subcellular Ca2+ signalling and reveals that many cell function are regulated by the microenvironment within small regions of the cytoplasm ('local control' concept). Here confocal imaging is used to measure and locate changes in [Ca2+]i on a subcellular level in response to receptor stimulation in visceral myocytes. We show that stimulation of muscarinic receptors in ileal myocytes with carbachol leading to activation of inositol 1,4,5-trisphosphate receptors (IP3Rs) accelerates the frequency of spontaneous calcium sparks (discharged via ryanodine receptors, RyRs) and gives rise to periodic propagating Ca2+ waves oscillating with a frequency similar to that of carbachol-activated cationic current oscillations. Furthermore, by combining the whole-cell patch clamp technique with simultaneous confocal imaging of [Ca2+]i in voltage-clamped vascular myocytes we demonstrate that calcium sparks may lead to the opening of either Ca2+-activated Cl- channels or Ca2+-activated K+ channels, and the discharge of a spontaneous transient inward current (STIC) or a spontaneous transient outward current (STOC), respectively.

Isolation and culture of rat superior mesenteric artery smooth muscle cells.

The structure and function of vascular smooth muscle cells have been extensively investigated with the aid of in vitro culture techniques. The majority of studies have utilized aortic tissue as the source of cells. We present here a method for isolating and culturing smooth muscle cells of the rat superior mesenteric artery, an elasto-muscular vessel that is structurally and functionally different from the aorta. Cells were isolated from partially digested explants and characterized by immunochemical and biochemical techniques. Unlike cultured fibroblasts, the cultured cells stained positive for smooth muscle specific actin. The cells also produced laminin and type IV collagen in culture. This method provides a means for the isolation of large numbers of viable smooth muscle cells from the superior mesenteric artery which can be propagated in culture for in vitro study.