Paracrine effects of hypoxic fibroblast-derived factors on the MPT-ROS threshold and viability of adult rat cardiac myocytes.

Cardiac fibroblasts contribute to multiple aspects of myocardial function and pathophysiology. The pathogenetic relevance of cytokine production by these cells under hypoxia, however, remains unexplored. With the use of an in vitro cell culture model, this study evaluated cytokine production by hypoxic cardiac fibroblasts and examined two distinct effects of hypoxic fibroblast-conditioned medium (HFCM) on cardiac myocytes and fibroblasts. Hypoxia caused a marked increase in the production of tumor necrosis factor (TNF)-alpha by cardiac fibroblasts. HFCM significantly enhanced the susceptibility of cardiac myocytes to reactive oxygen species (ROS)-induced mitochondrial permeability transition (MPT), determined by high-precision confocal line-scan imaging following controlled, photoexcitation-induced ROS production within individual mitochondria. Furthermore, exposure of cardiac myocytes to HFCM for 5 h led to loss of viability, as evidenced by change in morphology and annexin staining. HFCM also decreased DNA synthesis in cardiac fibroblasts. Normoxic fibroblast-conditioned medium spiked with TNF-alpha at 200 pg/ml, a concentration comparable to that in HFCM, promoted loss of myocyte viability and decreased DNA synthesis in cardiac fibroblasts. These effects of HFCM are similar to the reported effects of hypoxia per se on these cell types, showing that hypoxic fibroblast-derived factors may amplify the distinct effects of hypoxia on cardiac cells. Importantly, because both hypoxia and oxidant stress prevail in a setting of ischemia and reperfusion, the effects of soluble factors from hypoxic fibroblasts on the MPT-ROS threshold and viability of myocytes may represent a novel paracrine mechanism that could exacerbate ischemia-reperfusion injury to cardiomyocytes.

Endogenous nitric oxide mechanisms mediate the stretch dependence of Ca2+ release in cardiomyocytes.

Stretching of cardiac muscle modulates contraction through the enhancement of the Ca2+ transient, but how this occurs is still not known. We found that stretching of myocytes modulates the elementary Ca2+ release process from ryanodine-receptor Ca2+-release channels (RyRCs), Ca2+ sparks and the electrically stimulated Ca2+ transient. Stretching induces PtdIns-3-OH kinase (PI(3)K)-dependent phosphorylation of both Akt and the endothelial isoform of nitric oxide synthase (NOS), nitric oxide (NO) production, and a proportionate increase in Ca2+-spark frequency that is abolished by inhibiting NOS and PI(3)K. Exogenously generated NO reversibly increases Ca2+-spark frequency without cell stretching. We propose that myocyte NO produced by activation of the PI(3)K-Akt-endothelial NOS axis acts as a second messenger of stretch by enhancing RyRC activity, contributing to myocardial contractile activation.

Glucagon-like peptide-1 increases cAMP but fails to augment contraction in adult rat cardiac myocytes.

The gut hormone, glucagon-like peptide-1 (GLP-1), which is secreted in nanomolar amounts in response to nutrients in the intestinal lumen, exerts cAMP/protein kinase A-mediated insulinotropic actions in target endocrine tissues, but its actions in heart cells are unknown. GLP-1 (10 nmol/L) increased intracellular cAMP (from 5.7+/-0.5 to 13.1+/-0.12 pmol/mg protein) in rat cardiac myocytes. The effects of cAMP-doubling concentrations of both GLP-1 and isoproterenol (ISO, 10 nmol/L) on contraction amplitude, intracellular Ca(2+) transient (CaT), and pH(i) in indo-1 and seminaphthorhodafluor (SNARF)-1 loaded myocytes were compared. Whereas ISO caused a characteristic increase (above baseline) in contraction amplitude (160+/-34%) and CaT (70+/-5%), GLP-1 induced a significant decrease in contraction amplitude (-27+/-5%) with no change in the CaT after 20 minutes. Neither pertussis toxin treatment nor exposure to the cGMP-stimulated phosphodiesterase (PDE2) inhibitor erythro-9-(2-hydroxy-3-nonyl)adenine or the nonselective PDE inhibitor 3-isobutyl-1-methylxanthine nor the phosphatase inhibitors okadaic acid or calyculin A unmasked an ISO-mimicking response of GLP-1. In SNARF-1-loaded myocytes, however, both ISO and GLP-1 caused an intracellular acidosis (DeltapH(i) -0.09+/-0.02 and -0.08+/-0.03, respectively). The specific GLP-1 antagonist exendin 9-39 and the cAMP inhibitory analog Rp-8CPT-cAMPS inhibited both the GLP-1-induced intracellular acidosis and the negative contractile effect. We conclude that in contrast to beta-adrenergic signaling, GLP-1 increases cAMP but fails to augment contraction, suggesting the existence of functionally distinct adenylyl cyclase/cAMP/protein kinase A compartments, possibly determined by unique receptor signaling microdomains that are not controlled by pertussis toxin-sensitive G proteins or by enhanced local PDE or phosphatase activation. Furthermore, GLP-1 elicits a cAMP-dependent modest negative inotropic effect produced by a decrease in myofilament-Ca(2+) responsiveness probably resulting from intracellular acidification.

Paclitaxel stent coating inhibits neointimal hyperplasia at 4 weeks in a porcine model of coronary restenosis.

BACKGROUND: Despite limiting elastic recoil and late vascular remodeling after angioplasty, coronary stents remain vulnerable to restenosis, caused primarily by neointimal hyperplasia. Paclitaxel, a microtubule-stabilizing drug, has been shown to inhibit vascular smooth muscle cell migration and proliferation contributing to neointimal hyperplasia. We tested whether paclitaxel-coated coronary stents are effective at preventing neointimal proliferation in a porcine model of restenosis. METHODS AND RESULTS: Palmaz-Schatz stents were dip-coated with paclitaxel (0, 0.2, 15, or 187 microgram/stent) by immersion in ethanolic paclitaxel and evaporation of the solvent. Stents were deployed with mild oversizing in the left anterior descending coronary artery (LAD) of 41 minipigs. The treatment effect was assessed 4 weeks after stent implantation. The angiographic late loss index (mean luminal diameter) decreased with increasing paclitaxel dose (P<0.0028 by ANOVA), declining by 84.3% (from 0.352 to 0.055, P<0.05) at the highest level tested (187 microgram/stent versus control). Accompanying this change, the neointimal area decreased (by 39.5%, high-dose versus control; P<0.05) with increasing dose (P<0.040 by ANOVA), whereas the luminal area increased (by 90.4%, high-dose versus control; P<0.05) with escalating dose (P<0.0004 by ANOVA). Inflammatory cells were seen infrequently, and there were no cases of aneurysm or thrombosis. CONCLUSIONS: Paclitaxel-coated coronary stents produced a significant dose-dependent inhibition of neointimal hyperplasia and luminal encroachment in the pig LAD 28 days after implantation; later effects require further study. These results demonstrate the potential therapeutic benefit of paclitaxel-coated coronary stents in the prevention and treatment of human coronary restenosis.

The old heart: operating on the edge.

Excitation of cardiac cells is accompanied by Ca2+ influx which triggers a transient increase in cytosolic [Ca2+], (Cai), and contraction. While the amplitudes of the Cai transient and contraction increase with the extent of cell Ca2+ loading, excess Ca2+ loading leads to dysregulation of Ca2+ homeostasis, impaired contraction, arrhythmia and cell death. The cell Ca2+ load is determined by membrane structure and permeability characteristics, the intensity of stimuli that modulate Ca2+ influx or efflux via regulatory function of proteins within membranes, and reactive oxygen species (ROS), which affect both membrane structure and function. Cardiocytes of senescent hearts exhibit a reduced threshold for pathologic manifestations of excess Ca2+ loading during stimulation (physiologic or pharmacologic) that increases Ca2+ influx, e.g. in response to neurotransmitters, post-ischaemic reperfusion, or oxidative stress. Cell 'remodelling' is one cause of the relative Ca2+ intolerance of cardiocytes in the senescent heart; cells increase in size and changes occur in the amounts of proteins that regulate Ca2+ handling due, in part, to altered gene expression; another cause is a change in the composition of membranes in which Ca2+ regulatory proteins reside, e.g. an increase in membrane omega 6:omega 3 polyunsaturated fatty acids (PUFA); a third cause is an enhanced likelihood for intracellular generation of ROS. Each class of these determinants changes with ageing and reduces the threshold for Ca2+ overload to occur with the older heart. The risk of excess Ca2+ loading within the senescent heart can potentially be reduced by gene therapy to restore Ca2+ regulatory proteins, by diet to reverse the membrane omega 6:omega 3 PUFA imbalance, or by antioxidants.

Reactive oxygen species (ROS)-induced ROS release: a new phenomenon accompanying induction of the mitochondrial permeability transition in cardiac myocytes.

We sought to understand the relationship between reactive oxygen species (ROS) and the mitochondrial permeability transition (MPT) in cardiac myocytes based on the observation of increased ROS production at sites of spontaneously deenergized mitochondria. We devised a new model enabling incremental ROS accumulation in individual mitochondria in isolated cardiac myocytes via photoactivation of tetramethylrhodamine derivatives, which also served to report the mitochondrial transmembrane potential, DeltaPsi. This ROS accumulation reproducibly triggered abrupt (and sometimes reversible) mitochondrial depolarization. This phenomenon was ascribed to MPT induction because (a) bongkrekic acid prevented it and (b) mitochondria became permeable for calcein ( approximately 620 daltons) concurrently with depolarization. These photodynamically produced "triggering" ROS caused the MPT induction, as the ROS scavenger Trolox prevented it. The time required for triggering ROS to induce the MPT was dependent on intrinsic cellular ROS-scavenging redox mechanisms, particularly glutathione. MPT induction caused by triggering ROS coincided with a burst of mitochondrial ROS generation, as measured by dichlorofluorescein fluorescence, which we have termed mitochondrial "ROS-induced ROS release" (RIRR). This MPT induction/RIRR phenomenon in cardiac myocytes often occurred synchronously and reversibly among long chains of adjacent mitochondria demonstrating apparent cooperativity. The observed link between MPT and RIRR could be a fundamental phenomenon in mitochondrial and cell biology.

Glucagon-like peptide-1 does not mediate amylase release from AR42J cells.

In this study, AR42J pancreatic acinar cells were used to investigate if glucagon-like peptide-1 (GLP-1) or glucagon might influence amylase release and acinar cell function. We first confirmed the presence of GLP-1 receptors on AR42J cells by reverse trasncriptase-polymerase chain reaction (RT-PCR), Western blotting, and partial sequencing analysis. While cholecystokinin (CCK) increased amylase release from AR42J cells, GLP-1, alone or in the presence of CCK, had no effect on amylase release but both CCK and GLP-1 increased intracellular calcium. Similar to GLP-1, glucagon increased both cyclic adenosine monophosphate (cAMP) and intracellular calcium in AR42J cells but it actually decreased CCK-mediated amylase release (n = 20, P < 0.01). CCK stimulation resulted in an increase in tyrosine phosphorylation of several cellular proteins, unlike GLP-1 treatment, where no such increased phosphorylation was seen. Instead, GLP-1 decreased such protein phosphorylations. Genestein blocked CCK-induced phosphorylation events and amylase secretion while vanadate increased amylase secretion. These results provide evidence that tyrosine phosphorylation is necessary for amylase release and that signaling through GLP-1 receptors does not mediate amylase release in AR42J cells. J. Cell. Physiol. 181:470-478, 1999. Published 1999 Wiley-Liss, Inc.

Bcl-2 and Bcl-X(L) block thapsigargin-induced nitric oxide generation, c-Jun NH(2)-terminal kinase activity, and apoptosis.

The proteins Bcl-2 and Bcl-X(L) prevent apoptosis, but their mechanism of action is unclear. We examined the role of Bcl-2 and Bcl-X(L) in the regulation of cytosolic Ca(2+), nitric oxide production (NO), c-Jun NH(2)-terminal kinase (JNK) activation, and apoptosis in Jurkat T cells. Thapsigargin (TG), an inhibitor of the endoplasmic reticulum-associated Ca(2+) ATPase, was used to disrupt Ca(2+) homeostasis. TG acutely elevated intracellular free Ca(2+) and mitochondrial Ca(2+) levels and induced NO production and apoptosis in Jurkat cells transfected with vector (JT/Neo). Buffering of this Ca(2+) response with 1, 2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl) ester (BAPTA-AM) or inhibiting NO synthase activity with N(G)-nitro-L-arginine methyl ester hydrochloride (L-NAME) blocked TG-induced NO production and apoptosis in JT/Neo cells. By contrast, while TG produced comparable early changes in the Ca(2+) level (i.e., within 3 h) in Jurkat cells overexpressing Bcl-2 and Bcl-X(L) (JT/Bcl-2 or JT/Bcl-X(L)), NO production, late (36-h) Ca(2+) accumulation, and apoptosis were dramatically reduced compared to those in JT/Neo cells. Exposure of JT/Bcl-2 and JT/Bcl-X(L) cells to the NO donor, S-nitroso-N-acetylpenacillamine (SNAP) resulted in apoptosis comparable to that seen in JT/Neo cells. TG also activated the JNK pathway, which was blocked by L-NAME. Transient expression of a dominant negative mutant SEK1 (Lys-->Arg), an upstream kinase of JNK, prevented both TG-induced JNK activation and apoptosis. A dominant negative c-Jun mutant also reduced TG-induced apoptosis. Overexpression of Bcl-2 or Bcl-X(L) inhibited TG-induced loss in mitochondrial membrane potential, release of cytochrome c, and activation of caspase-3 and JNK. Inhibition of caspase-3 activation blocked TG-induced JNK activation, suggesting that JNK activation occurred downstream of caspase-3. Thus, TG-induced Ca(2+) release leads to NO generation followed by mitochondrial changes including cytochrome c release and caspase-3 activation. Caspase-3 activation leads to activation of the JNK pathway and apoptosis. In summary, Ca(2+)-dependent activation of NO production mediates apoptosis after TG exposure in JT/Neo cells. JT/Bcl-2 and JT/Bcl-X(L) cells are susceptible to NO-mediated apoptosis, but Bcl-2 and Bcl-X(L) protect the cells against TG-induced apoptosis by negatively regulating Ca(2+)-sensitive NO synthase activity or expression.

Activation of distinct cAMP-dependent and cGMP-dependent pathways by nitric oxide in cardiac myocytes.

Nitric oxide (NO) donors were recently shown to produce biphasic contractile effects in cardiac tissue, with augmentation at low NO levels and depression at high NO levels. We examined the subcellular mechanisms involved in the opposing effects of NO on cardiac contraction and investigated whether NO modulates contraction exclusively via guanylyl cyclase (GC) activation or whether some contribution occurs via cGMP/PKG-independent mechanisms, in indo 1-loaded adult cardiac myocytes. Whereas a high concentration of the NO donor S-nitroso-N-acetylpenicillamine (SNAP, 100 micromol/L) significantly attenuated contraction amplitude by 24.4+/-4.5% (without changing the Ca2+ transient or total cAMP), a low concentration of SNAP (1 micromol/L) significantly increased contraction amplitude (38+/-10%), Ca2+ transient (26+/-10%), and cAMP levels (from 6.2 to 8.5 pmol/mg of protein). The negative contractile response of 100 micromol/L SNAP was completely abolished in the presence of the specific blocker of PKG KT 5823 (1 micromol/L); the positive contractile response of 1 micromol/L SNAP persisted, despite the presence of the selective inhibitor of GC 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 10 micromol/L) alone, but was completely abolished in the presence of ODQ plus the specific inhibitory cAMP analog Rp-8-CPT-cAMPS (100 micromol/L), as well as by the NO scavenger oxyhemoglobin. Parallel experiments in cell suspensions showed significant increases in adenylyl cyclase (AC) activity at low concentrations (0.1 to 1 micromol/L) of SNAP (AC, 18% to 20% above basal activity). We conclude that NO can regulate both AC and GC in cardiac myocytes. High levels of NO induce large increases in cGMP and a negative inotropic effect mediated by a PKG-dependent reduction in myofilament responsiveness to Ca2+. Low levels of NO increase cAMP, at least in part, by a novel cGMP-independent activation of AC and induce a positive contractile response.

Enhanced expression of p53 and apoptosis induced by blockade of the vacuolar proton ATPase in cardiomyocytes.

Activation of the vacuolar proton ATPase (VPATPase) has been implicated in the prevention of apoptosis in neutrophils and adult cardiac myocytes. To determine the role of the VPATPase in apoptosis of cardiac myocytes, we used a potent and specific inhibitor of the VPATPase, bafilomycin A1. Bafilomycin A1 alone caused increased DNA laddering of genomic DNA and increased nuclear staining for fragmented DNA in neonatal cardiomyocyte apoptosis in a dose- and time-dependent manner. Intracellular acidification in cardiac myocytes was also observed after 18 h of bafilomycin A1 treatment. Accordingly, bafilomycin A1-treated myocytes also showed increased accumulation of p53 protein and p53-dependent transactivation of gene expression, including a persistent upregulation of p21/wild-type p53 activated fragment 1/cyclin kinase inhibitor protein-1 mRNA. The bafilomycin A1-induced increase in p53 protein levels was accompanied by a marked increase in p53 mRNA accumulation. In contrast, cardiac fibroblasts treated with bafilomycin A1 showed no change in p53 protein expression or pHi and did not undergo apoptosis even after 24 h of treatment. Our data suggest that blockade of the VPATPase induces apoptotic cell death of cardiac myocytes and that this may occur through a p53-mediated apoptotic pathway.

Inhibition of myocardial crossbridge cycling by hypoxic endothelial cells: a potential mechanism for matching oxygen supply and demand?

Previous studies have shown that cardiac endothelial cells release substances that influence myocardial contraction. Since PO2 is an important stimulus that modulates endothelial function, we investigated the effects of acute moderate hypoxia and reoxygenation on the release of cardioactive factors by endothelial cells. Endothelial cells cultured from several vascular beds were superfused with normoxic (equilibrated with room air; PO2, approximately 160 mm Hg) or hypoxic (PO2, 40 to 50 mm Hg) physiological buffer solution, and the superfusates were reequilibrated to a PO2 of approximately 160 mm Hg and then tested for their effects on various myocardial assays. Endothelial cell viability and buffer ionic composition were unaltered after the superfusion procedures. The superfusates of hypoxic endothelial cells induced rapid, potent, reversible inhibition of isolated cardiac myocyte contraction without reducing cytosolic Ca2+ transients. This activity was not lost after heating (95 degrees C) and was present in low molecular weight (Mr, <500) superfusate fractions. Hypoxic endothelial superfusate reduced unloaded shortening velocity of human skinned soleus muscle fibers. It markedly depressed in vitro actin motility over cardiac myosin and reduced the rate of actin-activated cardiac myosin ATPase activity but had no effect on corresponding smooth muscle myosin assays. Reoxygenation of hypoxic endothelial cells resulted in loss of this inhibitory activity. These data indicate that cultured endothelial cells respond to acute moderate hypoxia by releasing an unidentified substance(s) that inhibits myocardial crossbridge cycling, independent of Ca2+ or other second messenger signaling pathways. Such a mechanism could have important implications for the regulation of oxygen supply-demand balance in the heart and be relevant to conditions such as myocardial hibernation.

Mitogenic signaling mediated by oxidants in Ras-transformed fibroblasts.

NIH 3T3 fibroblasts stably transformed with a constitutively active isoform of p21(Ras), H-RasV12 (v-H-Ras or EJ-Ras), produced large amounts of the reactive oxygen species superoxide (.O2-). .O2- production was suppressed by the expression of dominant negative isoforms of Ras or Rac1, as well as by treatment with a farnesyltransferase inhibitor or with diphenylene iodonium, a flavoprotein inhibitor. The mitogenic activity of cells expressing H-RasV12 was inhibited by treatment with the chemical antioxidant N-acetyl-L-cysteine. Mitogen-activated protein kinase (MAPK) activity was decreased and c-Jun N-terminal kinase (JNK) was not activated in H-RasV12-transformed cells. Thus, H-RasV12-induced transformation can lead to the production of .O2- through one or more pathways involving a flavoprotein and Rac1. The implication of a reactive oxygen species, probably .O2-, as a mediator of Ras-induced cell cycle progression independent of MAPK and JNK suggests a possible mechanism for the effects of antioxidants against Ras-induced cellular transformation.

Redistribution of intracellular Ca2+ stores after beta-adrenergic stimulation of rat tail artery SMC.

beta-Adrenergic agonists induce the relaxation of vascular smooth muscle by a mechanism that activates the extrusion of Na+ and Ca2+ from the cell. A primary source of contractile Ca2+ resides in the sarcoplasmic reticulum (SR), which releases Ca2+ in response to vasoactive agents through inositol trisphosphate-mediated channels. To determine if smooth muscle relaxation induced by beta 2-adrenergic agonists involves the redistribution of intracellular Ca2+, we studied the effects of isoproterenol (Iso) on freshly isolated, single rat tail artery smooth muscle cells loaded with fura 2, using digital ratiometric fluorescence imaging. Stimulation with 1 microM phenylephrine (PE) or norepinephrine produced phasic and tonic increases in cytoplasmic intracellular Ca2+ concentration ([Ca2+]i) associated associated with cell shortening. Exposure to caffeine and to Ca2(+)-free solutions eliminated the phasic and tonic components, respectively, from the Ca2+ signal. Intermittent superfusion with PE or caffeine was used to evaluate SR Ca2+ stores after stimulation by Iso. Exposure to 1 microM Iso induced a time-dependent decrease in PE-activated peak and tonic [Ca2+]i without any change in resting [Ca2+]i. Intermittent stimulation with 10 mM caffeine revealed a similar decline in peak [Ca2+]i, indicating Iso-dependent depletion of SR Ca2+ stores. The Ca2+ that remained in the SR after prolonged exposure to Iso (30% of the pre-Iso level by 80 min at 22 degrees C) failed to elicit a contractile response. The cells, perfused with a Na(+)- and Ca2(+)-free medium to block Na+/ Ca2+ exchange, prevented depletion of the SR Ca2+ stores by Iso. We propose that Iso inhibits agonist-mediated Ca2+ influx through sarcolemmal Ca2+ channels and activates Ca2+ redistribution from storage sites in the SR to the extracellular compartment by a mechanism that involves Na+/Ca2+ exchange. These combined effects of Iso facilitate smooth muscle relaxation (and reduce vascular tonus) by reducing the increase in cytoplasmic Ca2+ evoked by vasoconstrictors.

Actomyosin interaction modulates resting length of unstimulated cardiac ventricular cells.

We sought to determine whether resting or diastolic cardiac myocyte length during low stimulation rates is regulated by myofilament interaction. Cytosolic Ca2+ concentration ([Ca2+]i, via indo 1 fluorescence) and length, in the presence and absence of 2,3-butanedione monoxime (BDM), a potent inhibitor of force production in striated muscle, were measured in rat and guinea pig cardiac myocytes at rest and after electrical stimulation. In tetanized cells BDM reduced steady contraction amplitudes for a given [Ca2+]i. In an actomyosin-sliding filament assay without Ca2+ or regulatory proteins, BDM decreased actin filament velocity along myosin. BDM increased both diastolic and resting cell lengths without changes in [Ca2+]i. The resting cell length also increased when [Ca2+]i was reduced by removing extracellular Ca2+, an effect further enhanced by BDM and by loading cells with the intracellular Ca2+ chelator, 1,2-bis(2-amino-phenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethylester. Thus myofilament interaction is present in cardiac cells, both at rest or during low rates of stimulation, and this myofilament interaction is regulated, in part, by the ambient [Ca2+]i.

Intracellular signaling pathways required for rat vascular smooth muscle cell migration. Interactions between basic fibroblast growth factor and platelet-derived growth factor.

Intracellular signaling pathways activated by both PDGF and basic fibroblast growth factor (bFGF) have been implicated in the migration of vascular smooth muscle cells (VSMC), a key step in the pathogenesis of many vascular diseases. We demonstrate here that, while bFGF is a weak chemoattractant for VSMCs, it is required for the PDGF-directed migration of VSMCs and the activation of calcium/calmodulin-dependent protein kinase II (CamKinase II), an intracellular event that we have previously shown to be important in the regulation of VSMC migration. Neutralizing antibodies to bFGF caused a dramatic reduction in the size of the intracellular calcium transient normally seen after PDGF stimulation and inhibited both PDGF-directed VSMC migration and CamKinase II activation. Partially restoring the calcium transient with ionomycin restored migration and CamKinase II activation as did the forced expression of a mutant CamKinase II that had been "locked" in the active state by site-directed mutagenesis. These results suggest that bFGF links PDGF receptor stimulation to changes in intracellular calcium and CamKinase II activation, reinforcing the central role played by CamKinase II in regulating VSMC migration.

Taxol inhibits neointimal smooth muscle cell accumulation after angioplasty in the rat.

Despite significant improvements in the primary success rate of the medical and surgical treatments for atherosclerotic disease, including angioplasty, bypass grafting, and endarterectomy, secondary failure due to late restenosis continues to occur in 30-50% of individuals. Restenosis and the later stages in atherosclerotic lesions are due to a complex series of fibroproliferative responses to vascular injury involving potent growth-regulatory molecules (such as platelet-derived growth factor and basic fibroblast growth factor) and resulting in vascular smooth muscle cell (VSMC) proliferation, migration, and neointimal accumulation. We show here, based on experiments with both taxol and deuterium oxide, that microtubules are necessary for VSMCs to undergo the multiple transformations contributing to the development of the neointimal fibroproliferative lesion. Taxol was found to interfere both with platelet-derived growth factor-stimulated VSMC migration and with VSMC migration and with VSMC proliferation, at nanomolar levels in vitro. In vivo, taxol prevented medial VSMC proliferation and the neointimal VSMC accumulation in the rat carotid artery after balloon dilatation and endothelial denudation injury. This effect occurred at plasma levels approximately two orders of magnitude lower than that used clinically to treat human malignancy (peak levels achieved in this model were approximately 50-60 nM). Taxol may therefore be of therapeutic value in preventing human restenosis with minimal toxicity.

Role of calcium/calmodulin-dependent protein kinase II in the regulation of vascular smooth muscle cell migration.

BACKGROUND: The migration of vascular smooth muscle cells (VSMCs) is a key event in the pathogenesis of many vascular diseases. We have previously shown that VSMC migration in response to platelet-derived growth factor (PDGF) is suppressed when cultured cells are growth-arrested and induced to differentiate. The present study was undertaken to elucidate the mechanism of this suppression. METHODS AND RESULTS: While both proliferating and growth-arrested VSMCs upregulated expression of the immediate early response genes, c-fos and JE (monocyte chemoattractant protein 1), growth-arrested VSMCs exhibited much smaller changes in intracellular calcium in response to PDGF and failed to activate the calcium/calmodulin-dependent protein kinase II (CaM kinase II). Blocking calcium-calmodulin interactions (50 mumol/L W7) or the activation of CaM kinase II (10 mumol/L KN62) in proliferating cells blocked their migration by more than 90%, whereas inhibition of protein kinase C activation had no significant effect on migration. Pretreatment of growth-arrested VSMCs with the calcium ionophore ionomycin resulted in an approximately 2.5-fold activation of CaM kinase II and increased migration of growth-arrested cells to 84 +/- 6% that of proliferating cells. These effects of ionomycin were blocked by inhibitors of CaM kinase II. Constitutively activated (ie, calcium/calmodulin-independent) CaM kinase II introduced by gene transfection into growth-arrested cells significantly increased migration toward PDGF from < 20% to > 70% that of proliferating cells. CONCLUSIONS: These results demonstrate that activation of CaM kinase II is required for VSMC migration, that its activation in response to PDGF is suppressed in growth-arrested VSMCs, and that this suppression of CaM kinase II activation is responsible, in large part, for the failure of growth-arrested VSMCs to migrate toward PDGF.

Novel method to alter length and load in isolated mammalian cardiac myocytes.

We have devised a novel technique enabling reversible gradations in the resting and contraction length of intact mammalian ventricular myocytes of up to 15-18% over slack length. Enzymatically isolated single cells are embedded in a transparent, elastic, cross-linked fibrin matrix, contained within a narrow elastic tube. Reversible gradations in cell length are produced via fibrin matrix stretch, produced by stretching the tube. Simultaneous measurement of cell length, edge motion, and indo 1 fluorescence during auxotonic contractions permits characterization of cell contractile function. Although force cannot be directly measured, the time integral of contractile force (i.e., relative contractile impulse, a contractile index that is independent of shortening constraints) is derived combining myocyte shortening and matrix loading. Relatively small degrees of myocyte stretch produce a lightly afterloaded model dominated by variations in preload in which there is parallel augmentation of shortening and contractile impulse (force) development. At higher degrees of stretch, significant afterloading is introduced, resulting in the development of an inverse relationship between shortening and impulse (approaching isometric conditions). Length-dependent Ca2+ myofilament activation and load-dependent relaxation are readily demonstrated in intact isolated mammalian ventricular myocytes.

8-bromo-cGMP reduces the myofilament response to Ca2+ in intact cardiac myocytes.

The role of cGMP in myocardial contraction is not established. Recent reports suggest that nitric oxide, released by endothelial cells or within myocytes, modifies myocardial contraction by raising cGMP. We studied the effects of 8-bromo-cGMP (8bcGMP, 50 mumol/L) on contraction (cell shortening) and simultaneous intracellular Ca2+ transients (indo 1 fluorescence ratio) in intact adult rat ventricular myocytes (0.5 Hz and 25 degrees C) 8bcGMP reduced myocyte twitch amplitude and time to peak shortening (-19.6 +/- 4.2% and -17.6 +/- 1.3%, respectively) and increased steady-state diastolic cell length (+0.6 +/- 0.1 microns, mean +/- SEM, n = 8; all P < .05) but had no effect on shortening velocity, systolic or diastolic fluorescence ratio, or time to peak fluorescence ratio (all P = NS). In 7 of 13 myocytes, this negative inotropic effect was preceded by a transient positive inotropic effect, with small increases in twitch amplitude, shortening velocity, and cytosolic Ca2+ transient. Analysis of 8bcGMP effects on both the dynamic and steady-state relation between cell shortening and intracellular Ca2+ (during twitch contraction and tetanic contraction, respectively) indicated reduction in the myofilament response to Ca2+ in all cases. These 8bcGMP effects were inhibited by KT5823 (1 mumol/L), an inhibitor of cGMP-dependent protein kinase, or by the presence of isoproterenol (3 nmol/L). 8bcGMP had no effect on cytosolic pH in cells (n = 4) loaded with the fluorescent probe carboxyseminaphthorhodafluor-1. These data indicate that cGMP may modulate myocardial relaxation and diastolic tone by reducing the relative myofilament response to Ca2+, probably via cGMP-dependent protein kinase.