Cholera toxin treatment of vascular smooth muscle cells decreases smooth muscle alpha-actin content and abolishes the platelet-derived growth factor-BB-stimulated DNA synthesis.

The second messenger cyclic AMP regulates diverse biological processes such as cell morphology and cell growth. We examined the role of the second messenger cyclic AMP on rat aortic vascular smooth muscle cell (VSMC) morphology and the intracellular transduction pathway mediated by platelet-derived growth factor beta-receptor (PDGF-Rbeta). The effect of PDGF-BB on VSMCs growth was assessed by [(3)H]-thymidine incorporation. Tyrosine phosphorylation of PDGF-Rbeta, PLC-gamma1, ERK1 and ERK2, p125(FAK) and paxillin as well as Sm alpha-actin was examined by the chemiluminescence Western blotting method. Actin mRNA level was quantitated by Northern blotting. Visualization of Sm alpha-actin filaments, paxillin and PDGF-Rbeta was performed by immunfluorescence microscopy. Cholera toxin (CTX; 10 nM) treatment lead to a large and sustained increase in the cyclic AMP concentration after 2 h which correlated with change of VSMC morphology including complete disruption of the Sm alpha-actin filament array and loss of focal adhesions. Treatment of VSMCs with CTX did not influence tyrosine phosphorylation of p125(FAK) and paxillin but decreased the content of a Sm alpha-actin protein. Maximal decrease of 70% was observed after 24 h of treatment. CTX also caused a 90% decrease of the actin mRNA level. CTX treatment completely abolished PDGF-BB stimulated DNA-synthesis although PDGF-Rbeta level and subcellular distribution and translocation was not altered. Furthermore CTX attenuated the PDGF-BB-induced tyrosine phosphorylation of the PDGF-Rbeta, PI 3'-K, PLC-gamma1 and ERK1/2 indicating an action of cyclic AMP on PDGF-beta receptor. We conclude that although cyclic AMP attenuates the PDGF-Rbeta mediated intracellular transduction pathway, an intact actin filament may be required for the PDGF-BB-induced DNA synthesis in VSMCs.

Renal proliferative and phenotypic changes in rats with two-kidney, one-clip Goldblatt hypertension.

Angiotensin II (AII) is a vasoconstrictive peptide with hypertrophic and mitogenic effects on many cell types. Previous studies have shown that in vivo administration of AII in rats results in proliferation of, and phenotypic changes in, many renal cell populations, but in doses also causing hypertension. Thus, it was not possible to differentiate nonhemodynamic from hypertensive effects of AII. Therefore, we studied rats with renin-dependent, AII-mediated hypertension (the two-kidney, one-clip Goldblatt model; mean systolic blood pressure 238 +/- 48 v 140 +/- 6 mm Hg in sham-operated controls). The unclipped kidneys, which were exposed to high blood pressure, developed significant glomerular and tubulointerstitial injury, tubulointerstitial cell proliferation, dense focal interstitial monocyte-macrophage influx, increased deposition of types I and IV collagen, as well as increased cellular expression of desmin and actin, in tubulointerstitial areas when examined at 11 weeks. In contrast, clipped kidneys, protected from hypertension but with high local renin expression, had minimal abnormalities. These studies suggest that in this model increased renin, and presumably AII, does not mediate significant proliferative or phenotypic changes in the kidney in the absence of hypertension at 11 weeks.

Structural alterations in vascular endothelial tight junctions in the course of their gradual degradation in vitro.

Rabbit aorta explants in organ culture maintained their endothelium as a confluent cell layer for 1-6 days. Depending on culture time, interendothelial tight junctions underwent gradual morphological changes in their substructure, as seen in freeze-fracture replicas. The formation of a P-face associated groove and concurrent confluence of tight junction particles on E-faces after 24 hr in vitro was followed by a rarefaction of particles and shortening of tight junctional strands. By day 6 in vitro, almost all tight junctions have disappeared. We interpret these findings as signs of a degradation of tight junctions in vitro, involving three different substructural components: a component facing the protoplasm, tight junction particles and a component facing the extracellular space. The degradation was inhibited by culturing under increased ambient pressure (910 mmHg).

The induction of smooth muscle cell proliferation in vitro using an organ culture system.

Agents which promote vascular smooth muscle cell (SMC) proliferation are still unknown and cannot be readily defined in vivo. To examine this problem, we have developed a technique to maintain arteries in culture for more than two weeks without loss of endothelium and without loss of contractility. These vessel segments were maintained in 30% calf serum, and yet SMC replication rate in the media was found to be below 0.1% per day. No loss of medial cells nor medial necrosis was observed. Gentle endothelial denudation did not cause intimal thickening. If, in addition to denudation, direct mechanical injury was applied, then the replication rate of intimal SMC was found to be 60% per day during the first week. These cells were confirmed to be of SMC origin using cell specific antibodies. Since SMC start to proliferate after mechanical injury and not after application of serum, we conclude in our experimental model that exogenous growth factors play a minor role in the process of induction of proliferation of these cells.

Electrical properties of the plasma membrane of microplasmodia of Physarum polycephalum.

Microplasmodia of Physarum polycephalum have been investigated by conventional electrophysiological techniques. In standard medium (30 mM K+, 4 mM Ca++, 3 mM Mg++, 18 mM citrate buffer, pH 4.7, 22 degrees C), the transmembrane potential difference Vm is around -100 mV and the membrane resistance about 0.25 omega m2. Vm is insensitive to light and changes of the Na+/K+ ratio in the medium. Without bivalent cations in the medium and/or in presence of metabolic inhibitors (CCCP, CN-, N3-), Vm drops to about 0 mV. Under normal conditions, Vm is very sensitive to external pH (pH0), displaying an almost Nernstian slope at pH0 = 3. However, when measured during metabolic inhibition, Vm shows no sensitivity to pH0 over the range 3 to 6, only rising (about 50 mV/pH) at pH0 = 6. Addition of glucose or sucrose (but not mannitol or sorbitol) causes rapid depolarization, which partially recovers over the next few minutes. Half-maximal peak depolarization (25 mV with glucose) was achieved with 1 mM of the sugar. Sugar-induced depolarization was insensitive to pH0. The results are discussed on the basis of Class-I models of charge transport across biomembranes (Hansen, Gradmann, Sanders and Slayman, 1981, J. Membrane Biol. 63:165-190). Three transport systems are characterized: 1) An electrogenic H+ extrusion pump with a stoichiometry of 2 H+ per metabolic energy equivalent. The deprotonated form of the pump seems to be negatively charged. 2) In addition to the passive K+ pathways, there is a passive H+ transport system; here the protonated form seems to be positively charged. 3) A tentative H+-sugar cotransport system operates far from thermodynamic equilibrium, carrying negative charge in its deprotonated states.

Intimal thickenings of jugular veins after application of a stimulus known to be sclerogenic in arteries.

The present study examined the intimal reactions of rabbit jugular veins to a stimulus known to elicit arteriosclerotic alteration in the artery wall. Repeated transmural electrical stimulation was applied to external jugular veins of both normo- and hypercholesterolaemic rabbits. Endothelial permeability, as well as changes in intimal architecture, were investigated by electron microscopy. Initially, the veins responded to electrical stimulation with an increased transendothelial transport of horseradish peroxidase (40,000 daltons). After application of the stimulation program for 4 weeks, intimal fibrous thickening (33%), cellular fibrous proliferation (50%), and organized mural thrombi were observed. The fibrous thickening was characterized by an abundance of connective tissue matrix and paucity of subendothelial cells. The cellular fibrous proliferate predominantly consisted of myocytes with few interspersed monocytes/macrophages and granulocytes. It resembled intimal plaques induced in carotid arteries by the same method. However, the venous thickenings showed limited size and a more pronounced fibrous response when compared with the arteriosclerotic lesions. The morphological similarities between the observed venous intimal thickenings and the different types of phlebosclerotic manifestations described in the literature, especially intimal proliferations in vein grafts, render the model of electrical stimulation suitable for the elucidation of underlying pathogenic mechanisms.

Regrowth of arterial endothelium. Denudation with minimal trauma leads to complete endothelial cell regrowth.

Endothelial regeneration in the rat carotid artery was investigated using two different techniques of denudation. With balloon catheter denudation, medial cell death occurred, and endothelial regrowth stopped after several weeks, leaving a large area devoid of endothelium. After denudation with a new technique that removed the endothelium without damaging the media complete endothelial regrowth was achieved. Acutely after this denudation, large platelet thrombi were present on the subendothelial surface of vessels denuded with the filament loop. In contrast, balloon catheter denuded arteries showed only a platelet monolayer on their luminal surface. Within the first few weeks after denudation with either technique the regenerating endothelial cells stained strongly for basic fibroblast growth factor. At later times when replication of endothelium had stopped, the balloon catheter denuded vessels did not stain with this antibody. After filament denudation endothelial cell replication remained high until regrowth was complete and intensive staining was observed in the regenerating endothelial cells at all times. No differences were seen in staining of smooth muscle cells for transforming growth factor-beta and fibronectin in either set of denuded vessels. Both groups showed transforming growth factor-beta to be located in the developing intima and especially on the apical surface of luminal smooth muscle cells. The surface of these luminal smooth muscle cells also stained with antibody to fibronectin. These data demonstrate that total regrowth of endothelium can occur over large denuded areas despite the presence of transforming growth factor-beta and fibronectin on these surfaces. Furthermore the ability of these endothelial cells to proliferate would appear to be dependent on the presence of basic fibroblast growth factor and on the severity of the trauma induced by denudation.

Factors controlling the development of arterial lesions after injury.

This review discusses the role of growth factors on the proliferation of smooth muscle cells (SMCs) in injured arteries. We have used a variety of procedures to injure rat carotid arteries and noted that removal of endothelium followed by platelet adherence does not always initiate SMC replication. Furthermore, thrombocytopenia did not reduce the early SMC replication induced by balloon catheter injury. Platelet-derived growth factor (PDGF) has been found to exert little effect on SMC replication but markedly influences the ability of SMCs to migrate to the intima. Basic fibroblast growth factor (bFGF) is a potent mitogen for SMCs in denuded arteries while having no effect on cells in control uninjured arteries. We have hypothesized that arterial injury leads to release of bFGF from injured SMCs and so stimulates cell replication. Rats were treated with antibodies to bFGF immediately before balloon injury, and this significantly reduced the SMC replication. These findings suggest that in vivo bFGF is an important mitogen for initiating SMC replication and that PDGF is important as a chemotactic for SMCs.

Angiotensin II-induced progression of neointimal thickening in the balloon-injured rat carotid artery is AT1 receptor mediated.

To investigate the relative importance of AT1 and AT2 receptors in angiotensin II (Ang II)-induced restimulation of neointimal smooth muscle cell (SMC) DNA synthesis and increased neointimal cross-sectional area (CSA), male Wistar rats were subcutaneously infused for 2 weeks with Ang II and losartan, an AT1 receptor antagonist, or Ang II and PD123319, an AT2 receptor antagonist, during the third and fourth week after balloon injury of the left common carotid artery. Concomitantly, all rats received 5-bromo-2'-deoxyuridine to label DNA-synthesizing SMCs. Neointimal CSAs and SMC DNA synthesis were compared with control groups that received Ang II, 0.9% NaCl, losartan, or PD123319. Systolic blood pressure (SBP) was measured at different times during the infusion. Ang II induced an increase in SBP that was significantly different from the SBP in the NaCl group. Infusion of Ang II together with losartan reduced the Ang II-induced increase in SBP to levels comparable with those obtained in the NaCl group. Infusion of Ang II+PD123319 caused an increase in SBP that was comparable with the increase in SBP of the Ang II group and significantly different from the SBP of the NaCl group. Infusion of losartan or PD123319 alone did not affect SBP. Ang II significantly enhanced neointimal CSA (47%, P < .05) compared with the control group infused with NaCl. Losartan significantly reduced Ang II-induced neointimal thickening (neointimal CSA, -37%, P < .05). Infusion of PD123319 together with Ang II did not affect Ang II-induced neointimal thickening. Losartan or PD123319 alone did not reduce neointimal thickening, since the neointimal CSAs in these groups did not differ from the neointimal CSA of the NaCl group. Comparable effects were found for SMC DNA synthesis in the neointima. Ang II infusion increased neointimal SMC DNA synthesis. Addition of losartan reduced the fraction of DNA-synthesizing neointimal SMCs from 23.7 +/- 2.1% in the Ang II group to 12.8 +/- 1.8% in the Ang II+losartan group, whereas the labeling fraction in the neointima remained 26.6 +/- 3.1% in the Ang II+PD123319 group. The labeling fractions in the neointimas of the groups that received losartan or PD123319 alone did not differ from the labeling fraction in the NaCl group. These data indicate that AT1 but not AT2 receptors mediate the progression of neointimal thickening induced by delayed application of Ang II in the injured left carotid artery in the rat. Furthermore, these data suggest that AT1 and AT2 receptors are not involved in the regulation of normal growth of a neointima in the third and fourth week after balloon injury.

Alpha 1-receptor antagonists urapidil and prazosin inhibit neointima formation in rat carotid artery induced by balloon catheter injury.

Sympathetic nerves and catecholamines seem to have a trophic influence on vascular smooth muscle cells in vivo. We therefore tested whether alpha 1-antagonists would inhibit proliferation in arterial smooth muscle in vivo. Smooth muscle cells were stimulated to form a neointima in rat carotid arteries after deendothelialization by means of a 2F embolectomy catheter. The development of intimal lesions was determined 14 days after injury. The size of the neointima was measured using two parameters. Intimal DNA content was estimated from 5-mm segments of carotid arteries after DNA extraction and staining with Hoechst-stain. The size of the neointima was determined morphometrically as intimal area in histological cross-sections. Prazosin and urapidil were given orally once per day. Urapidil-treated rats showed significant inhibition of neointima formation for both parameters in a dose-dependent fashion. For prazosin a significant reduction could only be observed if DNA content was considered.

Inhibition of leukocyte extravasation with a monoclonal antibody to CD18 during formation of experimental intimal thickening in rabbit carotid arteries.

In the rabbit model of electrically induced intimal thickening, the adherence processes of different leukocyte subsets as well as the functional significance of leukocyte invasion in the initial migration of smooth muscle cells (SMCs) into the intima were studied by using monoclonal antibody (MAb) 60.3 (directed to the leukocyte adherence glycoprotein CD18), a known potent inhibitor of leukocyte adhesive functions. In control carotid arteries exposed to two periods of electrical stimulation within 36 hours, leukocytes, including all granulocyte subsets, monocytes, and lymphocytes, invaded the cell-free subendothelium. Concomitantly, SMCs were observed to migrate from the media into the intima. In the MAb 60.3-treated rabbits, however, neutrophil emigration into the stimulated arteries was abolished, whereas mononuclear leukocyte accumulation in the intima was only partially inhibited, indicating a complete CD18-dependent mechanism for neutrophil extravasation and additional receptor-ligand systems for the emigration of mononuclear leukocytes. SMCs moved into the intima despite complete blockage of neutrophils and the reduced accumulation of mononuclear cells within the subendothelium after MAb administration. These results preclude neutrophils as initiators of SMC migration into the intima. The influence of mononuclear cells on the migratory behavior of SMCs in intimal thickening formation, however, needs further elucidation.

Mouse model of arterial injury.

In the present study, we established an injury model of the mouse carotid artery. Complete removal of the endothelium was achieved with a flexible wire. A platelet monolayer covered the denuded surface, and damage to underlying medial smooth muscle cells (SMCs) was detected. Injection of [3H]thymidine was used to determine the replication index for medial SMCs, which was found to be 1.6% at 2 days after denudation and 9.8% at 5 days. SMCs were observed in the intima by day 8 (replication index, 66%), and by 2 weeks the intimal lesion had a similar cell content as the media. In most animals, repair of the endothelial lining was complete 3 weeks after injury. The present model will allow us to use transgenic animals to address questions relevant to vascular biology and atherosclerosis.

Expression of smooth muscle myosin in relation to growth kinetics of cultured aortic smooth muscle cells.

The goal of this study is to quantify smooth muscle myosin (SMM) expression at the level of the individual cell and to ascertain whether SMM expression in cultured aortic smooth muscle cells is related to definite growth phases, and whether the initial seeding density affects growth or SMM staining. Rabbit aortic smooth muscle cells (SMCs) were harvested by enzyme digestion of aortic tissue and plated at low (100 cells cm-2), medium (1000 cells cm-2), and high (10,000 cells cm-2) densities. Independent of seeding density, the lag phase lasted 2 to 3 days and, at all three densities, the growth rate during the logarithmic growth phase was almost the same. However, the time, the number of population doubling needed to reach the plateau phase and the cell number in the plateau were influenced by the initial seeding density. Immunofluorescence staining with anti-smooth muscle myosin (ASMM) revealed intensive staining of striated and filamentous patterns in all cells during the lag and early logarithmic growth phases. During the late logarithmic growth phase, two subpopulations of cells appeared, one showing a positive and the other no reaction with SMM antiserum. The lowest relative number of cells which showed positive reactions with SMM antiserum was observed toward the end of the logarithmic growth phase. During the plateau phase, the SMM-positive subpopulation increased, amounting to about 60% of the total number of cells, independent of the seeding density. In terms of absolute numbers, the number of SMM-positive cells increased over the course of 21 days by factors of 13, 72, and 342 for high, medium, and low seeded cultures, respectively. We conclude that a SMC subpopulation can divide without loss of SMM and that some, but not all, cells which lose their SMM may possibly regain it in the postconfluent state.

Regulation of smooth muscle cell growth in injured artery.

The process of intimal thickening after arterial injury can be divided into several steps, namely, initiation of smooth muscle cell proliferation, migration, and further intimal proliferation and deposition of matrix. Factors controlling the initiation of proliferation include de-endothelialization and vessel distension but not platelet adherence. Migration and subsequent intimal proliferation are controlled by factors from platelets, re-endothelialization, and endogenously released gamma-interferon.

Kinetics of cellular proliferation after arterial injury. V. Role of acute distension in the induction of smooth muscle proliferation.

Arteries denuded by the passage of a balloon catheter demonstrate marked smooth muscle cell (SMC) proliferation, whereas arteries denuded by a fine wire do not. We examined the possibility that vessel distension in addition to endothelial loss accounted for the SMC proliferation in the balloon model. Segments of rat left carotid isolated by temporary ligatures were distended hydrostatically with tissue culture medium at 300 mm Hg pressure via an external carotid cannula. Animals received either continuous [3H]thymidine infusion (intraperitoneally, Alzet pump, Alz Corp., Palo Alza, California) for 14 days or pulsed with [3H]thymidine at 1, 2, 3, or 7 days, and SMC proliferation was assessed by autoradiography. Distended vessels demonstrated patchy denudation acutely and complete endothelial regeneration by 3 days. No SMC were present in the intima at later times. Nevertheless, cumulative SMC proliferation over 14 days was 21 +/- 3% (perfused but not distended control: 0.8 +/- 0.6%; partially de-endothelialized but not distended control: 0.3 +/- 0.1%). Pulse-labeling demonstrated a peak of SMC proliferation between 2 and 3 days after injury. These results suggest that acute distension can contribute to the induction of SMC proliferation after endothelial denudation.

Intimal lesion formation in rat carotid arteries after endothelial denudation in absence of medial injury.

Injury of an artery by passage of a balloon catheter causes both endothelial denudation and medial damage and produces a marked smooth muscle cell (SMC) proliferative response. In this study, the endothelium from rat carotid arteries was removed by use of a rotating loop of 5/0 monofilament suture (gentle denudation technique), which did not cause any detectable damage to the underlying medial cells but did cause platelet adherence. Expression of platelet-derived growth factor (PDGF) A-chain and PDGF receptor mRNA was comparable to that seen in ballooned carotids, but the medial SMC proliferative response to gentle denudation was markedly reduced when compared to that observed after balloon denudation (1.4% vs. 13.6%). Intimal lesions were only observed in those zones that remained denuded for more than 7 days. These results demonstrate that a denuding injury with no medial trauma is sufficient to induce intimal lesions and that the significantly higher proliferation seen in ballooned vessels might reflect a response of the medial cells to trauma that occurred during denudation.

Effects of H2O2 on membrane potential and [Ca2+]i of cultured rat arterial smooth muscle cells.

The effect of 1 mmol/l H2O2 was studied on the membrane potential and [Ca2+]i with microelectrodes and the fura-2 technique, respectively. H2O2 induced a biphasic increase in [Ca2+]i with a fast transient peak and a subsequent plateau. H2O2 also led to a biphasic hyperpolarization of the cells with a similar time course. This was followed by a slight depolarization after wash-out of H2O2. External Ca2+ free solutions and treatment with the Ca2+ ionophore A23187 (1 mumol/l) abolished the effect of H2O2 on [Ca2+]i and almost entirely reduced the effect on the membrane potential. Phenylephrine (10 mumol/l) or A23187 also induced very similar biphasic hyperpolarizations of the membrane as H2O2 which were fully reversible after wash-out. It is concluded that H2O2 hyperpolarizes the membrane by opening of Ca2+ dependent K+ channels.

T lymphocytes inhibit the vascular response to injury.

The proliferation of vascular smooth muscle cells is controlled by specific growth factors and cytokines acting in paracrine networks. Macrophage products such as the platelet-derived growth factor and interleukin 1 promote smooth muscle proliferation and are released in the arterial wall during atherosclerosis and repair processes. T lymphocytes are also present in vascular tissue, but their role in vascular growth control in vivo has been unclear. We now demonstrate that rats in which T lymphocytes have been eliminated by a monoclonal antibody develop larger proliferative arterial lesions after balloon-catheter injury. Larger lesions also develop in athymic rnu/rnu rats that lack T lymphocytes, when compared with rnu/+ littermates with normal T-cell levels. Finally, injection of the lymphokine interferon gamma inhibits smooth muscle proliferation and results in smaller lesions compared with controls injected with buffer alone. These results indicate that T lymphocytes modulate smooth muscle proliferation during vascular repair. We propose that T lymphocytes may play an important, immunologically nonspecific role in tissue repair processes.

Cytocontractile structures and proteins of smooth muscle cells during the formation of experimental lesions.

The time course of structural changes in vascular smooth muscle cells (SMC) was investigated during the formation of an experimental lesion in response to balloon injury. We compared the filamentous organization, evaluated by quantitative electron microscopy, with the cellular content of two representative cytocontractile proteins (myosin and tropomyosin) as assessed by immunofluorescence. We found that the changes peak between 7 and 14 days after injury and that they are visible both in the neointima and to a lesser extent in the inner media. While virtually all SMC are of a filament-rich phenotype in the undisturbed media, after balloon injury SMC migrated into the intima and about 90% of these latter cells were either of a organelle-rich or an intermediate phenotype, with the remaining 10% being of the filament-rich phenotype. In the inner media about 40% of cells were either of organelle-rich or intermediate phenotype. In contrast to these profound organizational changes of responding SMC, histochemistry revealed only a slight and probably transient decrease of the cellular content of myosin and tropomyosin at that time point. Twenty-eight days after injury the discrepancies between the content and the organization of cytocontractile proteins became more apparent. While virtually all SMC showed a homogeneous intensive staining with both antibodies, indistinguishable from the media SMC, the organization of cytoplasmic filaments had not totally recovered. Even though this morphological study does not permit conclusions to be drawn on the contractile function of the cells, it shows that both the organization and the content of cytocontractile protein have to be analyzed and compared for SMC changes to be evaluated during the formation of an experimental lesion.

Mechanism of inhibition of neointimal formation by the angiotensin-converting enzyme inhibitor cilazapril. A study in balloon catheter-injured rat carotid arteries.

We investigated the mechanism of inhibition of neointima formation by the angiotensin-covering enzyme the carotid artery. We looked for the effects of cilazapril on all phases of the response to injury, ie, on proliferation of smooth muscle cells (SMCs) in the media, their migration, their proliferation in the neointima, and their disposition of extracellular matrix in the neointima. Although treatment was discontinued after 2 weeks, the inhibitory effect of cilazapril on neointimal formation was evident even 52 weeks after injury. The amount of extracellular matrix deposited in the intima during cilazapril treatment was decreased by 20% 2 weeks after injury, but no effect was seen if tissues were analyzed at 4 or 52 weeks. [3H]Thymidine-labeled cells (pulse labeling as well as 14-day continuous labeling) showed a decrease in SMC labeling in the tunica medica by 50%, but no inhibition in the labeling indices was seen in the neointima. The fraction of unlabeled neointimal cells in the cilazapril-treated rats as judged from continuous labeling experiments was inhibited by 86%. Taken together, these data suggest an antiproliferative effect on medial SMCs and an inhibition of SMC migration into the intima by cilazapril. Since intimal extracellular matrix deposition was only delayed, the decrease in medial SMC proliferation and subsequent migration seems to be the main reason for the reduction of neointima formation.