Effect of tiplaxtinin (PAI-039), an orally bioavailable PAI-1 antagonist, in a rat model of thrombosis.

OBJECTIVE: To assess the antithrombotic and profibrinolytic effects of tiplaxtinin (PAI-039), an orally bioavailable antagonist of PAI-1, in rat models of thrombosis. METHODS AND RESULTS: Carotid artery and vena cava vascular injury was produced by application of FeCl3 and blood flow was monitored using ultrasonic technology. To assess efficacy in a thrombosis prevention paradigm, PAI-039 was administered orally 90 min before injury (1-30 mg kg(-1)). To assess efficacy in a thrombosis treatment paradigm, vascular injury and stable thrombus formation were followed 4 h later by recovery and PAI-039 administration. PAI-039 prevented carotid artery occlusion in 20, 68 and 60% of animals pretreated with 0.3, 1.0 and 3.0 mg kg(-1), respectively. Time to occlusive thrombosis was increased from 18.2 +/- 4.6 min in controls to 32.5 +/- 8.7 (P = ns), 46.1 +/- 7.0 (P < 0.05), and 41.6 +/- 11.3 min (P < 0.05) in the respective PAI-039 treatment groups. In the vena cava protocol, PAI-039 pretreatment significantly reduced thrombus weight at PAI-039 doses of 3, 10 and 30 mg kg(-1). When PAI-039 was dosed in a treatment paradigm 4 h after stable arterial and venous thrombosis, a significant reduction in thrombus weight was observed 24 h later at PAI-039 doses of 3, 10 and 30 mg kg(-1). PAI-039 (10, 30 and 100 mg kg(-1)) had no effect on platelet aggregation in response to ADP or collagen and was not associated with increased bleeding or prolonged prothrombin time. In animals bearing no vascular injury, PAI-039 had no effect on circulating, low-levels of PAI-1 activity. In contrast, circulating PAI-1 activity increased 5-fold following the induction of vascular injury, which was completely neutralized by PAI-039. CONCLUSIONS: PAI-039 exerts antithrombotic efficacy in rat models of arterial and venous vascular injury without effecting platelet aggregation.

GW3965, a synthetic liver X receptor (LXR) agonist, reduces angiotensin II-mediated pressor responses in Sprague-Dawley rats.

BACKGROUND AND PURPOSE: Liver X receptors (LXRs) activate genes that regulate lipid and cholesterol metabolism. LXR agonists were shown recently to also increase murine renin gene expression in vivo. To further examine a link between lipid metabolism, the renin-angiotensin-aldosterone-system and blood pressure regulation, we investigated the effect of a LXR agonist (GW3965) on angiotensin II (Ang II)-mediated vasoreactivity and vascular angiotensin II receptor (ATR) gene expression. EXPERIMENTAL APPROACH: Arterial blood pressure (BP) was measured during Ang II infusions (1.5 min duration; 0.001-3 microg kg(-1)) in pentobarbital-anesthetized male Sprague-Dawley rats (n = 6-9) after oral administration of GW3965 (10 mg kg(-1), q.d.) or vehicle for 7 - 15 days. Mesenteric arteries and plasma were collected to analyze ATR gene expression and to measure plasma renin activity (PRA) and lipid profile, respectively. KEY RESULTS: Basal mean arterial pressure (MAP) was similar between groups. GW3965 dosing blunted the vasopressor effect of Ang II, which was significantly different with the 0.3 and 3 microg kg(-1) doses. No difference in heart rate, PRA or lipid profile was observed between groups. A time-course indicated that ATR type 1 and 2 gene expression of GW3965-treated vs. vehicle-treated rats decreased by 50%, reaching significance for ATR type 2, but not for ATR type 1, at time-points coinciding with BP measurements. CONCLUSIONS AND IMPLICATIONS: GW3965 decreased Ang II-mediated vasopressor responses coincident with a trend toward reduced ATR gene expression, suggesting that LXR agonists could affect vascular reactivity.

Effect of pharmacologic plasminogen activator inhibitor-1 inhibition on cell motility and tumor angiogenesis.

BACKGROUND: Plasminogen activator inhibitor-1 (PAI-1) is integrally involved in tumorigenesis by impacting on both proteolytic activity and cell migration during angiogenesis. OBJECTIVES: We hypothesized that an orally active small molecule inhibitor of PAI-1 (PAI-039; tiplaxtinin) could affect smooth muscle cell (SMC) attachment and migration in vitro on a vitronectin matrix, and exhibit antiangiogenic activity in vivo. METHODS: In vitro assays were used to assess the mechanism of inhibition of PAI-1 by PAI-039 using wild-type PAI-1 in the presence or absence of vitronectin and wild-type PAI-1 and specific PAI-1 mutants in SMC adhesion and migration assays. An in vivo tumor angiogenesis model was used to assess the effect of PAI-039 administration on neovascularization in a Matrigel implant. RESULTS: PAI-039 dose-dependently inhibited soluble, but not vitronectin-bound, PAI-1. Cell adhesion assays using PAI-1 mutants unable to bind vitronectin (PAI-1K) or inactivate proteases (PAI-1R) further suggested that PAI-039 inactivated PAI-1 by binding near its vitronectin domain. In a tumor angiogenesis model, PAI-039 treatment of wild-type mice dose-dependently decreased hemoglobin concentration and endothelial cell staining within the Matrigel implant, indicating reduced angiogenesis, but exhibited no in vivo efficacy in PAI-1 null mice. CONCLUSIONS: Administration of an orally active PAI-1 inhibitor prevented angiogenesis in a Matrigel implant. The lack of activity of PAI-039 against wild-type PAI-1 bound to vitronectin and PAI-1K suggests PAI-039 binding near the vitronectin-binding site. Our studies further substantiate a role for PAI-1 in cellular motility and tumor angiogenesis, and suggest for the first time that these effects can be modulated pharmacologically.

Characterization and comparative evaluation of a structurally unique PAI-1 inhibitor exhibiting oral in-vivo efficacy.

Plasminogen activator inhibitor-1 (PAI-1) is the major physiological inhibitor of both tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA). Elevated levels of PAI-1 are associated with thrombosis and vascular disease, suggesting that high plasma PAI-1 may promote a hypercoagulable state by disrupting the natural balance between fibrinolysis and coagulation. In this study, we identify WAY-140312 as a structurally novel small molecule inactivator of PAI-1, compare its inhibitory activity with other previously identified small molecule inhibitors, and investigate the mechanism of inactivation of PAI-1 in the presence of both tPA and uPA. In an immunofunctional assay, WAY-140312 inhibited PAI-1 with an estimated inhibitory concentration (IC(50)) of 11.7 micro m, which was the lowest value obtained of the four different PAI-1 inactivators tested. Surface activity profiling indicated that the critical micelle concentration for WAY-140312 was 95.8 micro m, and that each inhibitor exhibited unique physical chemical properties. Using a sensitive direct activity assay, the IC(50) for WAY-140312 was similar when either tPA or uPA was used as the target protease. Immunoblot analysis demonstrated that WAY-140312 near the IC(50) inhibited the complex formation between either tPA or uPA and PAI-1. After oral administration, WAY-140312 exhibited 29% bioavailability with a plasma half-life of approximately 1 h. In an in-vivo model of vascular injury, a 10 mg kg(-1) oral dose of WAY-140312 was associated with improvement in arterial blood flow and reduction in venous thrombosis. Thus, WAY-140312 represents a structurally novel small molecule inhibitor of PAI-1, and is the first such molecule to exhibit efficacy in animal models of vascular disease following oral administration.

Autocrine regulation of human preadipocyte migration by plasminogen activator inhibitor-1.

One of the initial stages of adipogenesis is migration of preadipocytes of mesenchymal origin into cell clusters to form primitive fat organs. The serine protease inhibitor plasminogen activator inhibitor-1 (PAI-1) is synthesized and released from human adipose tissue ex vivo and regulates smooth muscle and endothelial cell migration in vitro, but its role in adipose tissue is not known. We investigated the role of PAI-1 in cultures of human preadipocytes from men and women of various ages and body mass indexes. Human preadipocytes expressed the messenger ribonucleic acid for PAI-1 and released significant quantities of PAI-1 protein into the medium. As PAI-1 regulates motility through the interaction of vitronectin with its receptor, the integrin alphaVbeta3, we identified this receptor in human preadipocytes. Flow cytometric analysis indicated that human preadipocytes express the vitronectin receptor alphaVbeta3 in a similar pattern as human umbilical vein endothelial cells. Functional studies indicated that active, but not latent, PAI-1 inhibited preadipocyte attachment to vitronectin with an IC(50) of 13.3 nmol/L, and preincubation of vitronectin-coated Transwells with active PAI-1 prevented preadipocyte migration. Vitronectin was identified in homogenates of the stromal-vascular fraction of human adipose tissue, but was absent from human adipocytes and cultured preadipocytes. These data indicate that human preadipocyte migration is regulated through the endogenous expression of PAI-1 and alphaVbeta3 integrin, a novel autocrine mechanism for potentially regulating cell cluster formation in adipogenesis.

Release of PAI-1 by human preadipocytes and adipocytes independent of insulin and IGF-1.

The effect of insulin and insulin-like growth factor-1 (IGF-1) on plasminogen activator inhibitor-1 (PAI-1) release from primary cultures of human preadipocytes and adipocytes has been investigated. Initial experiments measuring basal PAI-1 release (ng/ml) indicated variability between individual cultures. Using a novel technique for adipocyte quantitation, additional experiments were performed to determine PAI-1 release per cell, indicating a significant reduction with differentiation. Insulin and IGF-1 over a range of concentrations had no effect on PAI-1 release, and RT-PCR of PAI-1 mRNA following treatment with insulin and IGF-1 also indicated similar expression between treatments. The cultures did exhibit insulin-stimulated glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression and leptin synthesis following differentiation to the adipocyte phenotype. This is the first report of PAI-1 secretion by primary cultures of human preadipocytes and adipocytes, indicating PAI-1 release independent of insulin and IGF-1 and implicating other factors in the elevated plasma PAI-1 observed with insulin resistance.

Development of a human adipocyte synthetic polymer scaffold.

Because of the need for methods for successful transplantation of autologous fat, the differentiation of human preadipocytes on surgical mesh coated with various extracellular matrix components was investigated. Biopsy specimens of human adipose tissue were collected from seven different patients and were subjected to collagenase digestion and selective filtration, resulting in primary cultures of human preadipocytes. Fluortex monofilament-expanded polytetrafluoroethylene (52-/xm pore size) was as a template for coating with either human collagen, albumin, or fibronectin, followed by sodding with established cultures of human preadipocytes. Sodding efficiency on the different matrices was determined by trypsinization of attached cells at different time periods. Preadipocytes did not attach to uncoated polytetrafluoroethylene, but did attach to protein-coated mesh, and in a variable manner. Fibronectin-coating resulted in the highest efficiency of sodding, with differentiation of preadipocytes to adipocytes as assessed by scanning electron -microscopy and conventional Oil Red O staining. Similar results were achieved by using rat (n = 6) perirenal adipose tissue. This new method of adipocyte scaffolding may be used for improving soft-tissue augmentation and serving as a delivery system for growth factors important in wound healing.

Synthesis and secretion of plasminogen activator inhibitor-1 by human preadipocytes.

To further investigate the role of plasminogen activator inhibitor-1 (PAI-1) in adipose tissue physiology, the production and regulation of PAI-1 was determined in primary cultures of human preadipocytes. When expressed as production per cell and cultured under identical conditions, human preadipocytes from both visceral (omental) and sc depots of lean and obese individuals released significant, yet similar, amounts of PAI-1 protein into the conditioned medium. High steady-state PAI-1 messenger RNA (mRNA) concentrations were observed in visceral and sc preadipocytes, with the relative level of expression equivalent to beta-actin mRNA. Tumor necrosis factor alpha significantly decreased PAI-1 production in a concentration-dependent manner in both visceral and sc cultures, whereas transforming growth factor beta significantly elevated PAI-1 production, but only in sc preadipocytes from obese individuals. Addition of insulin had no effect on antigen levels in conditioned medium of preadipocyte cultures. Stimulation of the preadipocyte cultures with a defined medium resulted in differentiation to the adipocyte phenotype, as determined by flow cytometric analysis, verifying the cultures as human preadipocyte. These studies are the first to observe significant PAI-1 mRNA expression and protein production in primary cultures of a human adipose tissue cellular component, and they suggest that nascent adipocytes contribute significantly to the elevated plasma PAI-1 observed in obesity.

PDGFbeta receptor blockade inhibits intimal hyperplasia in the baboon.

BACKGROUND: We have evaluated the use of a mouse/human chimeric anti-platelet-derived growth factor-beta receptor antibody in combination with heparin to inhibit intimal hyperplasia in the saphenous artery of the baboon after balloon angioplasty. METHODS AND RESULTS: The study evaluated lesion development in sequential injuries made 28 days apart. Each animal received control treatment after the first injury and antibody/heparin therapy after the second injury to the contralateral artery. The antibody was administered by bolus intravenous injections (10 mg/kg) on study days 1, 4, 8, 15, and 22 and heparin coadministered by continuous intravenous infusion at a dose of 0.13 mg/kg per hour. Morphometric analysis of tissue sections showed a 53% decrease in intimal area after antibody/heparin treatment (P=0.005), corresponding to a 40% decrease in the intima-to-media ratio (P=0.005). Smooth muscle cell proliferation in the injured wall, measured at both 4 and 29 days after balloon injury, were similar in the control and antibody/heparin-treated animals. CONCLUSIONS: These data suggest that platelet-derived growth factor plays a key role in the development of intimal lesions at sites of acute vascular injury in the nonhuman primate.

Angiotensin II receptors in human preadipocytes: role in cell cycle regulation.

The role of angiotensin II (AII) in human preadipocyte physiology has been investigated in primary cultures from human adipose tissue. Receptor binding studies indicated that human preadipocytes express a high affinity AII binding site of the AT1 subtype, as binding of 125I-labeled [Sar1,Ile8]AII was rapid, saturable, and specific. As AII has previously been demonstrated to affect the cell cycle in adrenal and cardiac cells, the effect of AII on regulation of cycle progression was examined in human preadipocytes. Stimulation of preadipocytes with AII resulted in G1 phase progression of the cell cycle, as determined by flow cytometric analysis. AII treatment was associated with induction of expression of the messenger RNA for the cell cycle regulatory protein cyclin D1 in a dose-dependent manner. Pretreatment of cells with subtype-selective AT receptor ligands before AII stimulation indicated that the cyclin response was mediated via the AT1 receptor. The identity of the cells as preadipocyte was verified by culture in a defined differentiation medium, observing both leptin message expression and triglyceride accumulation by flow cytometry. These findings indicate that AII has early, receptor-mediated effects on cell cycle progression in human preadipocytes that may contribute to differentiation to the adipocyte phenotype.

Identification of estrogen receptor beta RNA in human breast and abdominal subcutaneous adipose tissue.

This study reports the initial observation of the novel estrogen receptor, ER beta, in human subcutaneous adipose tissue. Human adipose tissue was obtained from various anatomic sites including the subcutaneous depots of lipectomy patients (healthy controls), and from subcutaneous abdominal and breast depots of lean and obese women with breast cancer. ER beta mRNA expression, determined by reverse transcription and polymerase chain reaction (RT-PCR), was present in each adipose tissue sample examined. Cloning and sequencing of the PCR product confirmed its identity as ER beta. Separation of adipose tissue into component fractions indicated that ER beta was expressed in both adipocytes and the stroma-vasculature. Primary culture of human preadipocytes indicated that ER beta mRNA was present only after differentiation to the adipocyte phenotype. This novel observation of ER beta mRNA indicates that this receptor subtype may have a role in ER-mediated responses in human adipose tissue.

A review of the microcirculation of adipose tissue: anatomic, metabolic, and angiogenic perspectives.

Adipose tissue microcirculation is unique within the vascular system because of a capacity for this tissue to grow throughout most of adult life. A review of the microcirculation of adipose tissue has included a historical review of the early studies, which served as a foundation for later investigations on this topic, including basic hemodynamic measurements in mammalian adipose tissue. The various methods for measuring blood flow in white and brown adipose tissue are discussed with respect to studies of transport of substrates involved in adipose tissue metabolism. The role of innervation and vascular adrenergic receptors and the effects of diet and exercise on adipose tissue blood flow are also included. An in-depth analysis of the development of adipose tissue microvasculature indicates that angiogenesis often precedes adipogenesis. The clinical effects of hemodynamic adaptations to adipose tissue expansion are discussed in view of an epidemic increase in the prevalence of obesity and its co-morbidities. The recent discovery of sites of nuclear regulation of adipocyte differentiation, together with the identification of growth factors in adipose tissue, is an indication of the progress that is being made in the further understanding of molecular and cellular events that affect adipose tissue growth and, ultimately, adipose tissue microcirculation.

Distribution of angiotensin II receptors in rat and human adipocytes.

Angiotensin II (AII) receptor binding assays were performed in rat adipocytes from three separate anatomic depots. Fat cells were isolated by collagenase digestion, and plasma membranes were prepared from the epididymal, mesenteric, and retroperitoneal fat depots of male Sprague-Dawley rats at 100 days of age. Binding of 125I-labeled [Sar1,Ile8]AII was rapid, saturable, and specific in membranes from all depots, identifying a receptor with a similar affinity of approximately 1 nM. Site-associated differences in receptor number were observed, with epididymal and mesenteric fat cell membranes exhibiting significantly more receptors than retroperitoneal fat cells when binding was expressed per unit of membrane protein. When corrected for cell volume, the number of receptors per cell ranked epididymal > retroperitoneal > mesenteric. Inhibitory constants for the peptide agonists AII and AIII and the peptide antagonist [Sar1,Ala8]AII indicated similar affinities in all three depots. Because the receptor has been classified pharmacologically into two subtypes, the AT1 selective antagonist losartan, and the AT2 selective antagonist PD 123,319 were used to classify the adipocyte receptor, indicating an AT1 subtype with an affinity for losartan in the mesenteric and retroperitoneal adipocytes that was significantly greater than the epididymal. Similar studies were performed in adipocyte membranes obtained from human omental and subcutaneous adipose tissue, revealing the presence of an AII receptor in both depots with an affinity of approximately 10 nM for losartan. These data indicate site-specific differences in AII receptor number in fat cell membranes from rats and the existence of human adipocyte AII receptors, suggesting that the adipocyte is significant for the peripheral metabolism of components of the renin-angiotensin system.

Identification and characterization of angiotensin II receptors in rat epididymal adipocyte membranes.

To better understand the role of the mitogenic vasoactive peptide angiotensin II (AII) in growth and differentiation, we have investigated the existence of membrane receptors for this peptide in rat adipocytes. Following isolation of epididymal fat cells, membrane protein was removed and incubated with varying concentrations of 125I-AII with or without submicromolar concentrations of unlabeled AII. Binding of AII was highly specific, rapid, and reversible. Scatchard analysis indicated that adipocyte membranes contain a high-affinity AII receptor with a Kd of 0.90 nmol/L and a binding site concentration (Bmax) of 53.7 fmol/mg protein. Additional pharmacologic analyses resulted in a rank order potency for peptide agonists and antagonists similar to that reported for the vascular receptor. Determination of subtype specificity with selective organic compounds indicated that the epididymal adipocyte receptor was displaced at low concentrations of DuP753, a selective AT1 subtype antagonist. These studies have successfully identified and characterized a high-affinity membrane receptor for AII in fat cells, further establishing adipose tissue as a peripheral site containing regulatory components of the local renin-angiotensin system.

Transforming growth factor alpha and atrial natriuretic peptide in white adipose tissue depots in rats.

To detect the presence in adipose tissue of peptides known to affect tissue growth and to investigate potential regional differences, epididymal and perirenal adipose tissue depots from male Sprague-Dawley rats were separated into adipocyte and stroma-vascular fractions by collagenase digestion, sequential centrifugation and filtration. Identity and integrity of the fractions were demonstrated by light and electron microscopy, while dose-response curves for angiotensin-converting enzyme (ACE) were performed, revealing maintained functional capacity of the stroma-vascular fraction. ACE, atrial natriuretic peptide (ANP), and transforming growth factor-alpha (TGF-alpha) concentrations were significantly greater in epididymal than perirenal stroma-vascular tissue. Adipocyte fractions from both depots contained significant concentrations of ANP and TGF-alpha. There was no detectable ACE in the adipocyte fractions, indicating that no contaminating stromal-vascular cells were present in these fractions. These data show significant concentrations of peptides with effects on growth in subfractions of adipose tissue and demonstrate regional differences in concentrations between fat depots.

Relationship of cardiac hemodynamic and biochemical adaptations to mortality during long-term aortic constriction.

To determine the biochemical and hemodynamic responses to aortic ligation, and to assess the survival rate after the induction of hypertension, 90 normotensive rats were subjected to surgical constriction of the abdominal aorta. Mortality, left ventricular hemodynamics, myocardial biochemical assays, and plasma renin assays were determined 1 week, 1 month, 3 months, or 1 year later. Mortality was greatest between 1 week and 3 months after aortic ligation, during which plasma renin activity was significantly elevated. The rate of left ventricular pressure rise, contractile index, and myocardial alpha-adrenoceptor number were increased at 1 month, but were comparatively depressed at 3 months after the operation, suggesting that the heart was in failure at this time. At 1 year after ligation, hemodynamic and biochemical parameters continued toward normalization. Our data suggest that, in this rodent model, cardiac pump failure occurs through a combination of time-dependent, pressure-induced mechanical adaptations and myocardial biochemical changes that involve both the renin-angiotensin and sympathetic nervous systems. The observed relationship between mortality, myocardial hemodynamics, and biochemical parameters may be used for additional basic research investigations concerning the early periods of cardiac failure.

Hemodynamic and metabolic correlates in adipose tissue: pathophysiologic considerations.

Research efforts investigating the pathophysiology of adipose tissue have often focused separately on either the metabolic or cardiovascular components of an expanding fat mass. However, the growth and development of the fat cells and their vasculature are closely interrelated, a fact that has been established through more than a century of diverse studies of adipose tissue. Recently, the prevalence of obesity in the United States has stimulated investigations into the cardiovascular and metabolic correlates occurring with excessive lipid deposition and subsequent adipose tissue expansion. These investigations have resulted in conclusive evidence that, from a cardiovascular perspective, obesity results in an elevated blood volume and cardiac output, accompanied by an expansion of adipose water space, whereas from a metabolic aspect, the disease is characterized by adipocyte enlargement and associated alterations in metabolic pathways and hormonal responsiveness. Because these separate areas of research have independently shown interdepot differences in perfusion requirements and metabolic adaptations during the transition from the lean to obese state, adipocyte expansion may be partially dependent on the pattern of vascularity. This hypothesis is discussed by examining the integral relationship between the cardiovascular system and adipocyte metabolism, hopefully providing new insight into control of the pathophysiological processes of an expanding adipose organ.

Effect of experimental obesity and subsequent weight reduction upon circulating atrial natriuretic peptide.

The effect of obesity and weight reduction upon circulating concentrations of atrial natriuretic peptide was assessed in an experimental model of the disease. Obese rats weighing in excess of 750 g were compared with formerly obese animals subjected to a 15-week period of caloric restriction resulting in a 40% reduction in body weight. Mean adipocyte size was significantly reduced with weight loss, as was estimated body fat. Mean arterial blood pressure remained normotensive for both groups, but a significant reduction in heart rate was associated with weight reduction. Circulating atrial natriuretic peptide was significantly elevated in the lean rats, which also exhibited decreased plasma renin activity and a negative sodium balance. Analysis of heart to body weight ratios implied that an obesity-associated, volume-induced cardiac hypertrophy remained even after the normalization of body fat. These results suggest that the diuresis and natriuresis accompanying weight reduction may be facilitated by atrial natriuretic peptide, which was elevated in part due to a persistent left ventricular hypertrophy following the transition from the obese to lean condition.

Cardiovascular effects of intermittent drinking: assessment of a novel animal model of human alcoholism.

The development of a novel model of human alcoholism has involved the presentation of a 30% alcohol solution to Sprague-Dawley rats via a syringe-feeding needle apparatus. With twice daily intermittent drinking, rats consumed an equivalent of 7-8 g/kg body weight of alcohol, which represented 25% of total daily caloric intake. Alcohol was absorbed rapidly, as significant circulating concentrations were observed within 15 min of gavage, eventually peaking at approximately 200 mg% 1 h later. Hemodynamic recordings in the conscious state after a 10-week drinking program indicated a normotensive blood pressure at peak blood alcohol levels, yet a hypertensive response 24 h after the final drink at a time when blood alcohol was not detected. Alcoholic rats continued to gain weight in parallel with controls fed ad libitum throughout the study, and changes in cardiac size and indices of contractility were not affected by 10 weeks of intermittent drinking. Additionally, no histological evidence of cardiac muscle damage was observed in alcoholic animals. Our animal model closely resembles the clinical situation in terms of the pattern of alcohol consumption, circulating concentrations of alcohol and the percentage of caloric intake in the form of alcohol. The hemodynamic changes observed support the hypothesis that alcoholic hypertension may be a manifestation of withdrawal, as opposed to any direct pressor effect of alcohol itself.