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Objective Abnormal pulmonary vasculature directly affects the development and progression of congenital diaphragmatic hernia (CDH)-associated pulmonary hypertension (PH). Though overarching structural and cellular changes in CDH-affected pulmonary arteries have been documented, the precise role of the extracellular matrix (ECM) in the pulmonary artery (PA) pathophysiology remains undefined. Here, we quantify the structural, compositional, and mechanical CDH-induced changes in the main and distal PA ECM and investigate the efficacy of mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) as a therapy to ameliorate pathological vascular ECM changes. Methods Pregnant Sprague-Dawley rodents were administered nitrofen to induce CDH-affected pulmonary vasculature in the offspring. A portion of CDH-affected pups was treated with intravenous infusion of MSC-EVs (1 × 1010 /mL) upon birth. A suite of histological, mechanical, and transmission electron microscopic analyses were utilized to characterize the PA ECM. Results The CDH model main PA presented significantly altered characteristics-including greater vessel thickness, greater lysyl oxidase (LOX) expression, and a relatively lower ultimate tensile strength of 13.6 MPa compared to control tissue (25.1 MPa), suggesting that CDH incurs ECM structural disorganization. MSC-EV treatment demonstrated the potential to reverse CDH-related changes, particularly through rapid inhibition of ECM remodeling enzymes (LOX and MMP-9). Additionally, MSC-EV treatment bolstered structural aspects of the PA ECM and mitigated pathological disorganization as exhibited by increased medial wall thickness and stiffness that, while not significantly altered, trends away from CDH-affected tissue. Conclusions These data demonstrate notable ECM remodeling in the CDH pulmonary vasculature, along with the capacity of MSC-EVs to attenuate pathological ECM remodeling, identifying MSC-EVs as a potentially efficacious therapeutic for CDH-associated pulmonary hypertension.

This paper collates the pathological findings from initial published autopsy reports on 23 patients with coronavirus disease 2019 (COVID-19) from 5 centers in the United States of America, including 3 cases from Houston, Texas. Findings confirm that COVID-19 is a systemic disease with major involvement of the lungs and heart. Acute COVID-19 pneumonia has features of a distinctive acute interstitial pneumonia with a diffuse alveolar damage component, coupled with microvascular involvement with intra- and extravascular fibrin deposition and intravascular trapping of neutrophils, and, frequently, with formation of microthombi in arterioles. Major pulmonary thromboemboli with pulmonary infarcts and/or hemorrhage occurred in 5 of the 23 patients. Two of the Houston cases had interstitial pneumonia with diffuse alveolar damage pattern. One of the Houston cases had multiple bilateral segmental pulmonary thromboemboli with infarcts and hemorrhages coupled with, in nonhemorrhagic areas, a distinctive interstitial lymphocytic pneumonitis with intra-alveolar fibrin deposits and no hyaline membranes, possibly representing a transition form to acute fibrinous and organizing pneumonia. Multifocal acute injury of cardiac myocytes was frequently observed. Lymphocytic myocarditis was reported in 1 case. In addition to major pulmonary pathology, the 3 Houston cases had evidence of lymphocytic pericarditis, multifocal acute injury of cardiomyocytes without inflammatory cellular infiltrates, depletion of splenic white pulp, focal hepatocellular degeneration and rare glomerular capillary thrombosis. Each had evidence of chronic cardiac disease: hypertensive left ventricular hypertrophy (420 g heart), dilated cardiomyopathy (1070 g heart), and hypertrophic cardiomyopathy (670 g heart). All 3 subjects were obese (BMIs of 33.8, 51.65, and 35.2 Kg/m2). Overall, the autopsy findings support the concept that the pathogenesis of severe COVID-19 disease involves direct viral-induced injury of multiple organs, including heart and lungs, coupled with the consequences of a procoagulant state with coagulopathy.

Obesity is a widespread condition that is more prevalent in Western countries compared to others. Aortic atherosclerosis (AA) is a condition that frequently has been associated with obesity. An obesity paradox, where morbidly obese decedents had either no or minimal AA compared to nonobese decedents, recently has been described by some of us. The explanation for this almost counterintuitive paradox has yet to be determined, but a number of hypotheses were advanced, including hemodynamic factors producing aortic wall shear stress (WSS). The purpose of the present study was to determine if there was a relationship between AA and WSS, as determined by postmortem measurement of aortic wall diameters.Circumferences of the aorta at the levels of the ascending, thoracic and abdominal aorta were measured in 274 consecutive autopsies over 2-year period of time. AA was assessed using a previously described grading scale as either mild or severe. Circumferences were mathematically converted to diameters and WSS was calculated using the Hagen-Poiseuille formula. Two different methods to estimate cardiac output were used, both based on literature methods, one of which was body mass index (BMI) dependent, and the other BMI independent. Univariate and multivariable analyses of the relationship between WSS, age, BMI, gender, race and severity of AA were performed.Of the 274 decedents, 140 had mild and 134 had moderate to severe AA. BMI35 was associated with moderate to severe AA. WSS was inversely correlated with AA in all these segments of the aorta in each BMI subgroup with the exception of the ascending aorta for decedents with BMI ≤35 kg/mOur data demonstrate that WSS was unexpectedly lower in decedents with moderate and severe AA as compared to those with mild AA. This observation, which requires further investigations, was seen in all BMI ranges and was confirmed by 2 methods to calculate WSS.

Introduction/Objective Sex disparity is reported across all forms of cardiovascular diseases. Only few studies have focused on sex differences in thoracic aortic disease pathology. We aim to identify and understand sex differences in this patient group to bridge the knowledge gap and improve clinicopathologic outcomes. Methods/Case Report This is a retrospective analysis of 83 proximal thoracic aortic aneurysm and dissection (TAAD) cases treated at a single quaternary care center in 2019. Chart review was done for demographics. Consensus criteria (Stone JR et al. Cardiovasc Pathol 2015; 24:267-78; Halushka MK et al. Cardiovasc Pathol 2016; 25:247-57) and a scoring system (Waters KM et al. Cardiovasc Pathol 2017; 30:6-11) were used for pathology reporting. Clinical correlation was also made. Pearson’s chi-square test was used for statistical analysis. Results (if a Case Study enter NA) 83 patients (61 male and 22 female) were retrieved. Overall thoracic aortopathy was higher among males, accounting for 73.4% of individuals with TAAD. In a subgroup analysis, there was no sex difference in dissection, aortic root involvement, and bicuspid aortic valve (p>0.05). Genetic aortopathy was more prevalent in females than males (27.2% vs 9.8%, p=0.04) alongside early age at first aortic event (median age: 31y vs 52y). Histopathologically, females had frequent translamellar mucoid extracellular matrix accumulation (45.4% vs 22.9%, p=0.04), extensive (54.5% vs 27.8%, p=0.02) and severe (59% vs 34.4%, p=0.04) elastic fiber fragmentation, higher band like (9% vs 6.5%, p>0.05) plus extensive (13.6% vs 4.9%, p>0.05) smooth muscle nuclei loss, and extensive (13.6% vs 1.6%, p=0.01) plus dense (4.5% vs 1.6%, p>0.05) laminar medial collapse than males. Conclusion In our patient population, females have a lower prevalence of thoracic aortic disease treated with open repair. However, those who develop TAAD harbor a greater burden of wall pathology and probable worse outcomes. We recommend sex-based analysis of all research on thoracic aortic diseases.

Turmeric (Curcuma Longa) has a near 4000-year history of extensive medical use in South Asia. Its main physiologically active phytochemical is curcumin (diferuloylmethane), derived from the rhizome of turmeric. Curcumin is a hydrophobic polyphenol with a diketone moiety connecting two phenoxy rings. It is widely available, and exerts systemic and pleiotropic effects via several key mechanisms. Most famously, it is known to inhibit pro-inflammatory pathways such as PI3k/akt/NF-kB activation. It is also a potent antioxidant and free radical scavenger via a sequential proton loss electron transfer mechanism in ionizing solvents due to its extended conjugating ability across the entire molecule, and its ability to induce NRF-2. It has been implicated in the treatment of diseases ranging from asthma to various cancers, and is also a broad spectrum anti-microbial. COVID-19 is a novel beta-coronavirus that was declared a pandemic by the WHO in March, 2020. It is primarily a respiratory disorder, but it can spread hematogenously and effect many other organs such as the heart, nervous system, and kidneys. There is a significant intersection between the clinical manifestations of COVID-19 and curcumin’s therapeutic effects. In addition, curcumin has been shown to inhibit initial viral infectivity. Thus, there is potential for curcumin to safely both prevent and treat COVID-19 infection across the globe.

Immune checkpoint inhibitor (ICI)-induced myocarditis carries a poor prognosis and is not fully understood. Similar to lymphocytic myocarditis and acute cellular rejection in heart transplant, ICI-induced myocarditis requires immune suppressive strategies. We aimed to describe ICI-induced myocarditis by presenting findings of comprehensive cardiovascular evaluations and outcomes of patients following a therapeutic approach similar to autoimmune disorders or allograft transplant rejection, and to discuss the molecular basis of the benefits of immune modulation and statins in ICI-myocarditis. Three patients with ICI-induced myocarditis (2 with positive biopsies and 1 based on cardiac magnetic resonance imaging with negative biopsy) underwent a complete cardiovascular workup, including cardiac catheterization with endomyocardial biopsy. Treatment was with intravenous immunoglobulins (IVIG) and statins in all cases, with additional colchicine (2 cases) or hydroxychloroquine (1 case). Immunohistochemical analysis demonstrated varied subsets of T cells involved in the inflammatory response. Therapy with IVIG and statins led to symptom resolution and cardiac function normalization at 1-month follow-up in all patients. Cancer therapy was resumed in all patients. One patient expired 10 months after the myocarditis episode due to advanced malignancy; two patients were alive, free of heart failure symptoms and cancer progression, at 1-year follow-up, and 1 patient was rechallenged with ICI. We suggest that treatment with IVIG and statins may allow for a prompt resumption of anti-cancer therapy (including ICI) and improve outcomes.

A universal definition of myocardial infarction (UDMI) has been established, periodically updated, and refined over the past twenty years. The primary purpose of the UDMI is to bring uniformity and accuracy to clinical diagnosis. Herein, a review and analysis of the UDMI is presented with emphasis on clinicopathological correlation. Determination of the presence of myocardial injury is based on the detection of abnormal serum cardiac biomarkers, particularly cardiac troponin (cTn), and in the current fourth iteration of the UDMI, high sensitivity (hs)-cTn. Differentiation of myocardial infarction from other causes of myocardial injury requires the documentation of clinical evidence of myocardial ischemia. In this review, difficulties in applying the UDMI in actual practice are discussed, based on the experience and perspective of those of us who face these problems as part of our own practice of pathology. The complexity in application of the UDMI is highlighted by the presentation of five illustrative cases involving the differential diagnosis of myocardial injury and myocardial infarction due to atherothrombotic and nonatherothrombotic coronary artery disease. The cases include myocardial infarction due to severe coronary atherosclerosis, supply-demand mismatch, coronary artery dissection associated with an eosinophilic coronary periarteritis, and coronary thromboembolism, and a case with a differential diagnosis of myocarditis and myocardial infarction. These cases illustrate how pathological findings can contribute to more accurate application of the UDMI and how, when critically applied, the UDMI can be used to better characterize myocardial infarcts in clinical practice.

While the basic pathobiology of myocardial ischemic injury and reperfusion has been determined over the last 50 years, there are important, unresolved, or at least not completely elucidated, issues in the field. These include the relative contributions of different modes of cell injury and death to evolving myocardial infarcts; interactions of phenomena produced by reperfusion, including stunning and preconditioning; and potential new approaches for successfully combining adjuvant therapy with coronary artery opening. A model of myocardial ischemic and reperfusion injury is proposed involving the potential expression of apoptotic and oncotic pathways in the same perturbed cardiomyocytes. Promising new cardioprotective strategies for reducing lethal reperfusion injury are discussed, including ischemic postconditioning, activators of the reperfusion injury salvage kinase (RISK) pathway, inhibitors of protein kinase c-delta, and inhibitors of the mitochondrial membrane permeability transition pore (PTP). Major outstanding clinical challenges are also discussed, including the development of clinical care systems that can routinely deliver very timely coronary opening and reperfusion, perhaps combined with adjuvant therapy, and the development of strategies to retard adverse remodeling and congestive heart failure in patients with significant myocardial infarction and scarring, perhaps by refinements in stem cell therapy.

During post-natal maturation of the mammalian heart, proliferation of cardiomyocytes essentially ceases as cardiomyocytes withdraw from the cell cycle and develop blocks at the G0/G1 and G2/M transition phases of the cell cycle. As a result, the response of the myocardium to acute stress is limited to various forms of cardiomyocyte injury, which can be modified by preconditioning and reperfusion, whereas the response to chronic stress is dominated by cardiomyocyte hypertrophy and myocardial remodeling. Acute myocardial ischemia leads to injury and death of cardiomyocytes and nonmyocytic stromal cells by oncosis and apoptosis, and possibly by a hybrid form of cell death involving both pathways in the same ischemic cardiomyocytes. There is increasing evidence for a slow, ongoing turnover of cardiomyocytes in the normal heart involving death of cardiomyocytes and generation of new cardiomyocytes. This process appears to be accelerated and quantitatively increased as part of myocardial remodeling. Cardiomyocyte loss involves apoptosis, autophagy, and oncosis, which can occur simultaneously and involve different individual cardiomyocytes in the same heart undergoing remodeling. Mitotic figures in myocytic cells probably represent maturing progeny of stem cells in most cases. Mitosis of mature cardiomyocytes that have reentered the cell cycle appears to be a rare event. Thus, cardiomyocyte renewal likely is mediated primarily by endogenous cardiac stem cells and possibly by blood-born stem cells, but this biological phenomenon is limited in capacity. As a consequence, persistent stress leads to ongoing remodeling in which cardiomyocyte death exceeds cardiomyocyte renewal, resulting in progressive heart failure. Intense investigation currently is focused on cell-based therapies aimed at retarding cardiomyocyte death and promoting myocardial repair and possibly regeneration. Alteration of pathological remodeling holds promise for prevention and treatment of heart failure, which is currently a major cause of morbidity and mortality and a major public health problem. However, a deeper understanding of the fundamental biological processes is needed in order to make lasting advances in clinical therapeutics in the field.

Sepsis remains a common and serious complication of major burn injury and currently accounts for over 54% of deaths in burn patients. Burns have been associated with high levels of circulating proinflammatory cytokines and immunosuppression which promotes systemic inflammatory response syndrome (SIRS) and sepsis, for which no effective treatment is currently available. Defensins and cathelicidins, a family of cationic naturally occurring antimicrobial peptides, are considered important components of the innate immune system as they play a major role in the body’s defense by inhibiting several bacteria, fungi and enveloped viruses. Our prior studies using RT-PCR and fluorescence deconvolution microscopy suggest decreased expression of human β defensin 2 (HBD2) in burn wounds. In this study we have characterized cathelicidin (LL-37) protein levels using in representative skin samples of deep partial and full thickness burns and in unburned skin. Our results show that in unburned skin, the majority of LL-37 was located in the malphigian layer and was concentrated on the stratum corneum as well as colocalized with sweat ducts. In burned skin, in which the epidermis was destroyed, this pattern of LL-37 was absent. The surviving dermal and subcutaneous layers revealed LL-37 presence in very high concentrations colocalized with sweat duct epithelia. The result of these studies will contribute to an understanding of the role of antimicrobial peptides in the pathophysiology of burn injury, associated immunosuppression, and sepsis.

Exposure of neonatal rat cardiac myocytes to palmitate and glucose produces apoptosis as seen by cytochrome c release, caspase 3-like activation, DNA laddering, and poly(ADP-ribose) polymerase cleavage. The purpose of this study was to understand the role of reactive oxygen species in the initiation of programmed cell death by palmitate. We found that palmitate (but not oleate) produces inhibition of carnitine palmitoyltransferase I, accumulation of ceramide, and inhibition of electron transport complex III. These events are subsequent to cytochrome c release and loss of the mitochondrial membrane potential. No differences in H2O2 production or N-terminal c-Jun kinase phosphorylation were detected between myocytes incubated in palmitate and control myocytes (nonapoptotic) incubated in oleate. These results suggest that the palmitate-induced loss of the mitochondrial membrane potential is not associated with H2O2 synthesis and that a membrane potential is required to generate reactive oxygen species following ceramide inhibition of complex III.

Neonatal rat cardiac myocytes were treated with cytokines, with or without the nitric oxide synthase (NOS) inhibitors N-monomethyl-L-arginine (LNMMA) and N-nitro-L-arginine methyl ester (LNAME), and systolic and diastolic calcium levels were measured by fluorescence spectrophotometry and confocal microscopy. Time-dependent changes following interferon-gamma (IFN-gamma) treatment revealed a continuing increase in intracellular calcium, which was reduced with LNMMA, but not with LNAME. Increases in calcium also occurred with interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha), but not to the extent seen with IFN-gamma. Increased cyclic guanosine monophosphate (cGMP) was involved in the results described with short-term (2 hr) TNF-alpha and long-term (18 hr) IFN-gamma treatments. Short-term exposure to IFN-gamma produced an increase in cyclic adenosine monophosphate (cAMP) and also an initial increase in the myocyte-bearing rate, with calcium levels either (i) subsequently returning to control levels while maintaining a fast beating rate or (ii), retaining a high systolic calcium level, but beating at control rates. Treatment with both IL-1beta and IFN-gamma stabilized the beating rate of the cells on some occasions. Shortening of myocytes increased with isoproterenol and following treatment with IFN-gamma, while isoproterenol stimulation of IFN-gamma-treated cells revealed increased contractile activity after short, but not long, treatment. LNMMA, but not reduced the increased contractile response with short-term IFN-gamma treatment. Our findings suggest that TNF-alpha acts via a cGMP-dependent pathway, whereas the actions of IFN-gamma involve adenylate cyclase, and possibly a NO-forming mechanism and cGMP pathway as well. It is also apparent that the two NO inhibitors function via different mechanisms or that LNMMA has a direct effect on the calcium-signaling pathway.

Fatty acids are the preferred substrate of ischemic, reperfused myocardium and may account for the decreased cardiac efficiency during aerobic recovery. Neonatal cardiac myocytes in culture respond to hypoxia/serum-and glucose-free medium by a slow decline in ATP which reverses upon oxygenation. This model was employed to examine whether carnitine palmitoyltransferase I (CPT-I) modulates high rates of β-oxidation following oxygen deprivation. After 5 h of hypoxia, ATP levels decline to 30% control values and CPT- I activity is significantly stimulated in hypoxic myocytes with no alteration in cellular carnitine content or in the release of the mitochondrial matrix marker, citrate synthase. This stimulation was attributed to an increase in the affinity of hypoxic CPT-I for carnitine, suggesting that the liver CPT-I isoform is more dominant following hypoxia. However, there was no alteration in hypoxic CPT-I inhibition by malonyl-CoA. DNP-etomoxiryl-CoA, a specific inhibitor of the liver CPT-I isoform, uncovered identical Michaelis kinetics of the muscle isoform in control and hypoxic myocytes with activation of the liver isoform. Northern blotting did not reveal any change in the relative abundance of mRNA for the liver vs. the muscle CPT-I isoforms. The tyrosine phosphatase inhibitor, pervanadate, reversed the hypoxia-induced activation of CPT-I and returned the affinity of cardiac CPT-I for carnitine to control. Reoxygenation was also associated with a return of CPT-I activity to control levels. The data demonstrate that CPT-I is activated upon ATP depletion. Lower enzyme activities are present in control and reoxygenated cells where ATP is abundant or when phosphatases are inhibited. This is the first suggestion that phosphorylation may modulate the activity of the liver CPT-I isoform in heart. (Mol Cell Biochem 180: 163–170, 1998)

This study investigates the hypothesis that inflammatory cytokines, interleukin (IL)-1alpha IL-1beta, and tumor necrosis factor (TNF), influence cardiac function by affecting calcium homeostasis and that this effect is mediated by the beta-adrenergic-adenylate cyclase system. After 4 days in culture, neonatal rat ventricular myocytes were treated with cytokines (10 ng/ml) for short (2 h) or longer (18 h) times. Myocyte calcium, contractility, and adenylate cyclase were measured under each condition. Anticipated stepwise increases in adenylate cyclase and intracellular calcium were found in controls (non-cytokine-treated) with 10(-7) M isoproterenol, 10(-7) M isoproterenol + 0.1 mM guanosine triphosphate, and 10(-9) M forskolin. Cells in the presence of cytokine for 2 h show increased basal calcium levels but no changes in adenylate cyclase activities, and isoproterenol fails to elevate adenylate cyclase levels or affect contractile shortening. After long-term treatment with IL-1beta or TNF, but not IL-1alpha, the significantly elevated levels of basal systolic calcium remain, and isoproterenol increases adenylate cyclase activity, unlike after short exposure. Forskolin maximally activates adenylate cyclase following both short- and long-term incubation, but the stepwise increase in activity is blunted following prolonged exposure. Thus short-term cytokine treatment blocks the adrenergic receptor-mediated increases in adenosine 3',5'-cyclic monophosphate, dissociating adenylate cyclase activation from cytokine-mediated increases in cell calcium, whereas longer treatment apparently produces direct affects on adenylate cyclase. Time-dependent differences in contractile response were found with IL-1alpha at 2 h and TNF at 18 h, implying that myofibrillar responsiveness to increased cytoplasmic calcium is dependent on both cytokine species and exposure time.

Patients with COL3A1 mutations that impede secretion of type III collagen into matrix develop wounds that heal poorly following cutaneous injury. They are also predisposed to vascular complications like catastrophic rupture or dissection of cervical arteries and stroke. We sought to determine if low production of type III collagen would also interrupt arterial thrombus resolution, a process resembling wound healing, and whether defects contribute to risk of dissection. We injured cervical elastic arteries in mice by ligation of the left common carotid, halting proximal blood flow. Strikingly, injured arteries from Col3a1 +/- mice develop thrombi over three weeks that resist resolution more often than those in wild-type littermates ( p =.002). The unresolved thrombi in Col3a1 +/- arteries also retain a higher burden of macrophages ( p =.043) and proliferative α-actin-positive cells ( p =.034), while mutant arteries display vascular channels within the media ( p =.015) that are partially lined with endothelial cells, consistent with residual neoangiogenesis. At two weeks, burdens of macrophages ( p =.009), proliferative cells ( p =.028), and vascular channels ( p =.019) are also higher in Col3a1 +/- arteries, despite actively resolving thrombi of equal length between the two groups ( p =.338), suggesting that neoangiogenesis results specifically from lower Col3a1 dose. Treating injured mice with rapamycin halts thrombus resolution in the early inflammatory phase of both genotypes and normalizes the incidence of vascular channels in Col3a1 +/- arteries at three weeks ( p =1.00), suggesting that vascular channels result secondary to thrombus resolution and not from structural weakness in type III collagen deficient arteries. Patient-derived COL3A1 mutant myofibroblasts in 3D culture models of fibrin remodeling accumulate less extracellular type I collagen following TGFβ1 stimulation but persistently upregulate expression of Acta2 , relative to control cells whose Acta2 expression levels peak and subside as collagen accumulates by seven days. These data implicate dysregulated thrombus remodeling in response to injury that increases the risk for medial neoangiogenesis, which may also increase the risk for dissection in affected arteries after injury.

We describe a patient who underwent a successful heart and kidney transplant for light-chain amyloidosis. She had an excellent hematologic response to bortezomib/dexamethasone therapy. Follow-up therapy with lenalidomide was started, and the patient quickly had a fatal allograft rejection of the heart and kidney. We present evidence to support the theory that lenalidomide, a known immunomodulator, may have stimulated the immune system and precipitated the fatal rejection episode.

The mitochondrial carnitine palmitoyltransferase (CPT) system is composed of two proteins, CPT-I and -II, which, together with carnitine acylcarnitine translocase, are involved in the transport of fatty acids into the mitochondrial matrix for beta-oxidation. In the liver, CPT-I and its inhibition by malonyl-CoA are sensitive to hormonal (10(-9) M) levels of insulin; however, a similar effect of insulin on heart CPT is controversial. In cultured neonatal rat cardiac myocytes, tissue culture concentrations (1.7 microM) of insulin increase CPT and cytochrome oxidase activities as well as mitochondrial protein synthesis, suggesting that a growth mechanism may be involved. Because insulin at high concentrations may interact with the insulin-like growth factor (IGF-I) receptor, the consequences of insulin's action on heart cells in culture may be mediated through the IGF pathway. Consistent with an IGF-mediated pathway for the effect of insulin, incorporation of radioactivity into immunoprecipitated CPT-II from insulin-treated cardiac myocytes is dramatically increased over control cells. The amount of immunoreactive CPT-I is also increased in insulin-treated cells. Moreover, an IGF-I analogue that inhibits the autophosphorylation of the IGF-I receptor blunts the insulin-mediated increase in CPT-I and -II activities by > 70%. At low physiologically relevant concentrations (10 ng/ml), IGF-I significantly increases the activities of both CPT-I and -II, and the IGF-I analogue eliminates the IGF-I response. This is the first study to suggest involvement of the IGF-I pathway in the regulation of mitochondrial CPT synthesis and activities in the heart.

Incubation of cultured neonatal rat cardiomyocytes in hypoxic conditions, mimicking the deprivation of O2 which occurs during in situ myocardial ischemia, leads to a progressive change in cardiomyocytes cytoskeletal components. Confocal scanning laser immunofluorescence microscopy (CSLIM) reveals that the typical striated costameric distribution of vinculin gradually disappears to be replaced by circular, vinculin-containing sarcolemmal rosettes. There is little change in distribution of vinculin in the focal adhesions or in the intercalated disks. This cytoskeletal alteration, like that observed in virally transformed fibroblasts and phorbol ester-treated skeletal myoblasts, is inhibited by genistein, a tyrosine kinase inhibitor. Increased exposure to hypoxic conditions also produces an increase in a 92-kDa collagenase which is immunolocalized only to cardiomyocytes. As with the rosette formation, genistein also inhibits the increased expression of the 92-kDa collagenase. We suggest that this cytoskeletal change with attendant release of 92 kDa collagenase may represent a defensive mechanism on the part of the cardiomyocyte to reduce damage by reducing the cellular coupling to the extracellular collagenous matrix, thereby lessening the stresses imposed by contractile forces.

HHV-6-infizierte Zellkulturlinien (HSB 2 T-Zellen; HDLM 2-Hodgkin-Zellen) sowie Biopsieproben von HHV-6-positiven Hodgkin(HD)- und Kikuchi-Lymphomen (KL) wurden immunhistologisch auf eine Expression der Genprodukte ras, bcl-2 und p 53 untersucht und die Ergebnisse korreliert mit Markern der Zellproliferation und mit Zeichen apoptotischen Zelltods. Die Ergebnisse der Zellkulturuntersuchungen sind eingeschrankt beurteilbar, da etablierte Zellinien bereits transformiert sind. Dennoch zeigen die Zellen mit geringerer Apoptoserate (HDLM 2) eine 10 fach geringere p 53-Expression und eine um die Halfte verminderte PCNA-Anfarbung der Zellen. In den Biopsien des starker nekrotisierenden KL war lediglich 1/6 bis 1/20 der ras-Expression des HD nachweisbar, p 53 fehlte ganzlich, wahrend bcl-2 deutlich gegenuber dem HD gesteigert war. PCNA-positive Zellen waren beim HD etwa 3 mal so haufig, wie beim KL. Es fanden sich keine signifikanten Unterschiede in der Virus-DNS oder Antigenexpression bei den verschiedenen untersuchten Zellen. Die Ergebnisse lassen vermuten, das weitere exogene Faktoren wie Zytokine und Wachstumsfaktoren in den Regelmechanismus fur virusinduzierte Zellproliferation und Apoptose eingreifen, und das neben der Apoptose andere Formen der viralen Zellyse vorkommen.

Clopidogrel is a thienopyridine derivative and a potent inhibitor of ADP-induced platelet aggregation. We compared clopidogrel with aspirin as an adjunctive treatment to tissue-type plasminogen activator (t-PA) for thrombolysis and reocclusion. Thrombosis was induced in coronary arteries of 32 dogs by injuring the endothelium with an electric charge. Coronary blood flow velocity was monitored by a pulsed Doppler flow probe placed around the artery. After the artery had been occluded by a thrombus for 3 continuous hours, each animal was given one of the following intravenous treatments: 1) t-PA (80 micrograms/kg + 8 micrograms.kg-1.h-1) and heparin (200 U/kg) (group 1, n = 7); 2) t-PA, heparin, and aspirin (5 mg/kg) (group 2, n = 8); 3) t-PA, heparin, and clopidogrel (5 mg/kg) (group 3, n = 9); and 4) t-PA, heparin, and clopidogrel (10 mg/kg + 2.5 mg.kg-1.h-1) (group 4, n = 8). After treatment, thrombolysis developed in 45 +/- 12 min in group 1, 39 +/- 10 min in group 2, 39 +/- 10 min in group 3, and 27 +/- 10 min in group 4 (compared with group 1, P > 0.05). After thrombolysis, reocclusion occurred in 5 of 5 dogs in group 1 and 7 of 7 in group 2, but only 2 of 7 in group 3 and none of 7 in group 4 (compared with groups 1 and 2, P < 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

An understanding of the mechanism of malonyl-CoA interaction with carnitine palmitoyltransferase (CPT-I) in isolated mitochondria is complicated by membrane fragmentation and CPT-II exposure. Using cultured neonatal rat cardiac myocytes, as in situ model was developed to measure CPT-I. In the cardiac cells treated with 5 microM digitonin, CPT-II contamination of CPT activity is 0.62% as quantitated by citrate synthase activity present in damaged myocytes under assay conditions. Moreover, the sensitivity of myocyte CPT-I to malonyl-CoA, its substrate preference for decanoyl-CoA and the affinity of CPT-I for l-carnitine (0.19 mM) are comparable with similar measurements published for isolated cardiac mitochondrial membranes. There is no evidence in the cells for contamination of CPT-I activities by extramitochondrial sources, in particular, the sarcoplasmic reticulum (SR). The presence of carnitine octanoyltransferase (COT) is not detected either in the cells or in preparations of adult SR from which COT is subsequently isolated. With these control measurements, the inhibition kinetics of CPT-I in the cardiac cells in situ maintains a partial competitive pattern which is more pronounced with decanoyl-CoA than with palmitoyl-CoA as substrate. The presence of a malonyl-CoA/long chain acyl-CoA binding site on CPT-I, distinct from the inhibitory site, has previously been proposed. Existence of this binding region is consistent with partial inhibition kinetics so that malonyl-CoA at this site could modify the CPT-high-affinity malonyl-CoA inhibitory interaction, producing acylcarnitine even at high malonyl-CoA concentrations in the cell. These findings may help to explain, in part, the inability to suppress completely beta-oxidation in the heart where malonyl-CoA may be 50 to 100 times the estimated values of its Ki.

Von Willebrand factor and platelet membrane glycoprotein Ib receptors interact to mediate platelet adhesion and thrombogenesis in stenosed and endothelium-injured arteries. We wished to determine whether blocking glycoprotein Ib receptors with a recombinant von Willibrand factor binding domain (VCL) increases the time required for thrombus formation after injury to the coronary arteries. We also wished to determine whether, after thrombolysis with tissue plasminogen activator (TPA), VCL delays or protects against coronary artery reocclusion. Twenty-seven dogs were treated with either saline, VCL, or aspirin before thrombosis was induced in their coronary arteries by electrical injury. The time from injury to the formation of occlusive thrombi was significantly greater with VCL (70 +/- 10 minutes) and aspirin (69 +/- 20 minutes) than with saline (18 +/- 3 minutes, P < .001 and P < .05). Thrombosis was induced in 30 other dogs that then received thrombolytic treatment in four groups. Our major finding was that coronary artery reocclusion occurred in 72 +/- 11 minutes after treatment with TPA (80 micrograms/kg + 8 micrograms.kg-1.min-1) and heparin (200 U/kg) (n = 7); in 142 +/- 24 minutes after TPA, heparin, and VCL (4 mg/kg + 2 mg.kg-1.h-1) (n = 7) (compared with TPA and heparin, P < .05); in 74 +/- 13 minutes after TPA, heparin, and aspirin (5 mg/kg) (n = 8); and in 173 +/- 8 minutes after TPA, heparin, VCL, and aspirin (n = 8) (compared with TPA and heparin, P < .001). Thus, VCL increases the length of time required for thrombus formation in coronary arteries, and, when given with TPA and heparin, delays coronary artery reocclusion more effectively than aspirin.

Damage to the cardiac myocyte sarcolemma following any of several pathological insults such as ischemia (anoxia) alone or followed by reperfusion (reoxygenation), is most apparent as progressive sarcolemmal blebbing, an event attributed by many investigators to a disruption in the underlying cytoskeletal scaffolding. Scanning electron microscopic observation of tissue cultured rat neonatal cardiomyocytes indicates that exposure of these cells to the toxic aldehyde 4-hydroxynonenal (4-HNE), a free radical-induced, lipid peroxidation product, results in the appearance of sarcolemmal blebs, whose ultimate rupture leads to cell death. Indirect immunofluorescent localization of a number of cytoskeletal components following exposure to 4-HNE reveals damage to several, but not all, key cytoskeletal elements, most notably microtubules, vinculin-containing costameres, and intermediate filaments. The exact mechanism underlying the selective disruption of these proteins cannot be ascertained at this time. Colocalization of actin indicated that whereas elements of the cytoskeleton were disrupted by increasing length of exposure to 4-HNE, neither the striated appearance of the myofibrils nor the lateral register of neighboring myofibrils was altered. Monitoring systolic and diastolic levels of intracellular calcium ([Ca2+]i) indicated that increases in [Ca2+]i occurred after considerable cytoskeletal changes had already taken place, suggesting that damage to the cytoskeleton, at least in early phases of exposure to 4-HNE, does not involve Ca(2+)-dependent proteases. However, 4-HNE-induced cytoskeletal alterations coincide with the appearance of, and therefore suggest linkage to, sarcolemmal blebs in cardiac myocytes. Although free radicals produced by reperfusion or reoxygenation of ischemic tissue have been implicated in cellular damage, these studies represent the first evidence linking cardiomyocyte sarcolemmal damage to cytoskeletal disruption produced by a free radical product.

A canine model with cyclic flow variations (CFVs) in stenosed and endothelium-injured coronary arteries was used to examine the role of active oxygen species in platelet aggregation in vivo. We studied 90 anesthetized dogs in which the pericardial cavity was opened and the heart was exposed. The velocity of blood flow in the left anterior descending coronary artery (LAD) was monitored by a pulsed Doppler flow probe. In 67 dogs, the LADs were stenosed by applying external constrictors at the site where the endothelium was mechanically injured. CFVs developed in all 67 dogs. Treatment with the antioxidants recombinant human copper-zinc superoxide dismutase (r-h-CuZnSOD), recombinant human manganese superoxide dismutase (r-h-MnSOD), and catalase eliminated platelet aggregation-associated coronary CFVs in 63%, 62%, and 64% of animals, respectively. Intravenous infusion of epinephrine restored CFVs in most dogs. Ketanserin, a serotonin (5-hydroxytryptamine2) receptor antagonist, abolished epinephrine-restored CFVs and eliminated CFVs in dogs in which CFVs had not been eliminated by free radical scavengers. In an additional 23 dogs, the LADs were stenosed but not mechanically injured. For control studies, saline was infused into the LADs of 5 dogs. Xanthine/xanthine oxidase was infused into the LADs of 8 dogs and induced CFVs in 4. Hydrogen peroxide was infused into the other 10 dogs and induced CFVs in 9. Histological analysis of the coronary artery revealed that the intima was significantly injured by the infusion. In ex vivo platelet aggregation studies, the in vivo treatment with r-h-CuZnSOD, r-h-MnSOD, and catalase significantly inhibited platelet aggregation induced by platelet-activating factor. Thus, active oxygen species are involved in mediating platelet aggregation and cyclic flow variations in stenosed and endothelium-injured canine coronary arteries in vivo.

BACKGROUND Platelet activating factor (PAF) is a phospholipid released upon stimulation by a variety of cells and has been implicated in several pathophysiological events such as asthma and inflammatory diseases. However, although the ability to aggregate platelets in vitro was the first biological activity ascribed to PAF, its role in contributing to the in vivo formation of arterial thrombi has not been thoroughly clarified. METHODS AND RESULTS Intravascular platelet aggregation was initiated in two different animal models of arterial stenosis and endothelial injury. An external constrictor was positioned around rabbit carotid arteries and canine coronary arteries. After placement of the constrictor, a typical pattern of flow developed in the stenotic vessels. This pattern of flow, characterized by progressive reductions of carotid or coronary blood flow followed by spontaneous or induced restorations of flow (cyclic flow variations, CFVs), is related to recurrent platelet aggregation at the site of the stenosis followed by dislodgment of the thrombus. After observing CFVs for 30 minutes, BN52021 (up to 1.2 mg/kg), a potent and selective PAF antagonist, was given intravenously to rabbits (n = 12) and dogs (n = 10). BN52021 completely inhibited CFVs in 10 of 12 rabbits, whereas it was relatively ineffective in abolishing CFVs in dogs (only 2 of 10 animals inhibited). This different effect of BN52021 was not explained by too small a dose of the drug to achieve a complete blockade of PAF receptors in dogs, since ex vivo platelet aggregation was completely inhibited in both rabbits and dogs in response to exogenous PAF at concentrations up to 10(-5) mol/L. In a second group of 10 dogs, the hypothesis that PAF may become an important mediator of CFVs in dogs only several hours after endothelial injury was tested. After 30 minutes of baseline CFVs, these animals received a bolus of BN52021 up to 1.2 mg/kg. After this treatment, CFVs were completely abolished in 2 of 10 animals. The remaining 8 dogs were followed for an additional 8-hour period, at the end of which a second bolus of BN52021 was given. At this time, BN52021 was effective, as CFVs were abolished in 6 of 8 animals. These effects of BN52021 at 8 hours were not the consequence of a cumulative dose of the compound, since ex vivo platelet aggregation in response to PAF returned to baseline values immediately before administering the second dose. To identify possible sources of PAF other than aggregating platelets at the site of arterial stenosis, dogs in a third group were killed after 30 minutes (n = 7) and after 8 hours (n = 8) of CFVs. Histological sections of the stenotic coronary artery showed a marked leukocyte infiltration in these arterial segments after 8 hours of CFVs, whereas sections from dogs killed after 30 minutes showed only moderate or no infiltration. CONCLUSIONS These data demonstrate that PAF plays an important role as a mediator of platelet aggregation in vivo in rabbits and dogs. In the canine model, PAF appears to become more important after leukocyte infiltration of the arterial wall, as it may contribute to initiating enough platelet activation to lead to cyclic flow variations at sites of arterial stenosis and endothelial injury. Data from the present study suggest that PAF antagonists may be used as antiplatelet agents.

BACKGROUND The efficacy of thrombolytic therapy in treating patients with acute myocardial infarction is limited by failure to achieve reperfusion in some patients, by the prolonged time required to achieve reperfusion, and by reocclusion of some coronary arteries. We designed this study to examine the effect of combined inhibition of thrombin and thromboxane synthesis and blockade of thromboxane A2 receptors in addition to tissue-type plasminogen activator (t-PA) on thrombolysis and reocclusion in an experimental canine model with coronary thrombosis. METHODS AND RESULTS Blood flow velocity in the left anterior descending coronary artery (LAD) of 32 anesthetized mongrel dogs was monitored by a pulsed Doppler flow probe. Coronary thrombosis was induced by applying electrical stimulation to the LAD at the site where an external constrictor was used to narrow the artery. Three hours after the formation of occlusive thrombus, animals were randomly assigned to receive one of the following: 1) t-PA (80 micrograms/kg + 8 micrograms.kg-1.min-1 i.v.) and saline; 2) t-PA and hirulog, a hirudin-based synthetic peptide and specific thrombin inhibitor (2 mg/kg + 2 mg.kg-1.hr-1 i.v.); 3) t-PA and ridogrel, a combined thromboxane A2 synthetase inhibitor and receptor antagonist (5 mg/kg + 2.5 mg.kg-1.hr-1 i.v.); or 4) t-PA, hirulog, and ridogrel. Reperfusion developed in 14% (one of seven) of dogs treated with t-PA alone at an average of 86 +/- 4 minutes after treatment, in 78% (seven of nine) of dogs treated with t-PA plus hirulog at 53 +/- 11 minutes, in 13% (one of eight) of dogs treated with t-PA plus ridogrel at 85 +/- 5 minutes, and in 88% (seven of eight) of dogs treated with t-PA, hirulog, and ridogrel at 37 +/- 10 minutes (comparison of the frequency of and the time to reperfusion, both p < 0.01). Among the dogs with reestablished coronary blood flow, reocclusion developed in the one treated with t-PA alone at 36 minutes after reperfusion, in seven of the seven treated with t-PA plus hirulog at 66 +/- 15 minutes, and in two of the seven treated with t-PA, hirulog, and ridogrel at 151 +/- 21 minutes (comparison of the frequency of and time to reocclusion, both p < 0.05). Reocclusion was not detected in the one dog treated with t-PA and ridogrel or in the other five dogs treated with t-PA, hirulog, and ridogrel within 180 minutes after reperfusion. Hirulog prolonged and maintained activated clotting times at a level twice that of baseline values. Hirulog inhibited ex vivo platelet aggregation induced by thrombin, and ridogrel inhibited platelet aggregation induced by U46619, a thromboxane mimetic. CONCLUSIONS Inhibition of thrombin in addition to treatment with t-PA enhances thrombolysis. A combination of inhibition of thrombin and thromboxane synthetase and blockade of thromboxane A2 receptor enhances thrombolysis and delays or may prevent reocclusion of the recanalized coronary arteries.

This study characterizes the sequential alterations of, and relations between, multiple electrolytes in cytoplasm, mitochondria, and whole cells during hypoxia and on reoxygenation in isolated neonatal rat ventricular myocytes. Subcellular electrolyte content and distribution were measured by electron probe x-ray microanalysis, membrane phospholipid degradation by tritiated arachidonic acid release, and cell morphology by electron microscopy. At 1-2 hours of hypoxia, the myocyte population showed a loss of cytoplasmic potassium, magnesium, and chlorine without alteration of cytoplasmic sodium or calcium. Mitochondria showed increased potassium with unchanged magnesium content. There was no morphological evidence of cell injury or tritiated arachidonic acid release. At 3-5 hours of hypoxia, the myocyte population showed a further loss of cytoplasmic potassium and magnesium and an increase in cytoplasmic sodium, chlorine, and calcium. At a single-cell level, the increase in cytoplasmic sodium preceded the increase in cytoplasmic calcium. Mitochondria showed increased sodium and chlorine and decreased magnesium before increased calcium content; potassium loss was manifest only at 5 hours of hypoxia. At 3-5 hours of hypoxia, there was also tritiated arachidonic acid release and morphological evidence of cell injury. Reoxygenation for 1 hour after 5 hours of hypoxia partially reversed the mean alterations of all electrolytes, except calcium, in the cytoplasm of the myocyte population, whereas analysis was required at a single-cell level to show a partial reversal in calcium levels in cytoplasm of reoxygenated cells. Reoxygenation for 1 hour after 5 hours of hypoxia partially reversed the mean alterations of all electrolytes, including calcium, in the mitochondria of the myocyte population. Recovery of potassium in the cytoplasm correlated with reduction of mitochondrial calcium content on reoxygenation and best predicted recovery of cellular homeostasis of sodium, chlorine, magnesium, and calcium. This study demonstrates that in this experimental model of hypoxia 1) initial losses of cytoplasmic potassium and magnesium occur in the absence of cell injury; 2) increases of sodium, chlorine, and calcium occur in association with cell injury, with sodium increasing before calcium; 3) membrane phospholipid degradation and electrolyte derangement, including increased calcium, may contribute to reversible and irreversible phases of cell injury; 4) analysis of calcium at a subcompartmental level and at a single-cell level is required to correlate reduction of calcium on reoxygenation with recovery of cell homeostasis; 5) reduction of calcium content in mitochondria may predict recovery of cell homeostasis; and 6) recovery of potassium on reoxygenation best predicts recovery of cell membrane function and cell homeostasis.

The objectives of this study were to test the hypotheses that thrombin inhibitors 1) enhance tissue-type plasminogen activator (t-PA)-induced coronary thrombolysis and 2) prevent or delay coronary artery reocclusion. Seventy-one dogs developed occlusive coronary thrombi after introducing a copper coil into the left anterior descending coronary artery (LAD). Coronary blood flows were monitored by an externally positioned pulsed Doppler flow probe. t-PA was given with or without heparin at different times after LAD occlusions. In some experiments, hirugen, a synthetic hirudin-based peptide and specific thrombin inhibitor, was given as 4 mg/kg i.v. bolus and 3 mg.kg-1.h-1 i.v. infusion at 30 min after LAD occlusion with t-PA and a bolus of heparin. Thrombolysis times were significantly shorter in t-PA- and heparin-treated dogs than in dogs treated with t-PA alone. Reocclusion times were significantly longer in t-PA- and heparin-treated dogs than in dogs treated with t-PA alone. Continuous heparin infusions prolonged reocclusion times to greater than 180 min in all treated dogs. The addition of hirugen to t-PA plus one bolus heparin prolonged reocclusion times to 90 +/- 6 min in dogs with 30-min thrombi. Thus heparin enhances t-PA-induced thrombolysis and delays reocclusion. Addition of a specific thrombin inhibitor, such as hirugen, to heparin enhances its effect on delaying reocclusion.

Prostacyclin is a critical mediator of structure and function in the pulmonary circulation, causing both the inhibition of vascular smooth muscle growth and vasodilation via the stimulation of adenylate cyclase. To examine the potential role of alterations in prostacyclin production or mechanism of action in chronic hypoxic pulmonary hypertension, we determined the effects of prolonged (7 d) in vivo hypoxia on in vitro prostacyclin synthesis and mediation of adenylate cyclase activity in rat main pulmonary arteries. In control arteries prostacyclin production exceeded that of prostaglandin (PG) E2 by 25-fold, with 42% originating from the endothelium. Studies utilizing indomethacin revealed that endogenous prostaglandins mediate at least 69% of basal adenylate cyclase activity. Prostacyclin-stimulated enzyme activity was enhanced by exogenous GTP, indicating that this is a receptor-mediated process involving G protein amplification. Comparable dose-related responses to prostacyclin and PGE2 suggest that these agents may activate a common receptor. After 7 d of in vivo hypoxia there was a 2.7-fold increase in in vitro prostacyclin production, with equivalent increases in synthesis in the endothelium and vascular smooth muscle. However, despite this increase there was no change in basal adenylate cyclase activity, and this was associated with attenuated sensitivity of the enzyme to prostacyclin stimulation. Concomitant diminution of the response to beta-adrenergic stimulation, with previously-demonstrated beta receptor downregulation and unaltered postreceptor-mediated activity, suggests that the blunted response to prostacyclin is due to receptor downregulation. Parallel studies of the thoracic aorta indicated that these changes are specific to the pulmonary artery. It is postulated that attenuation of the response of adenylate cyclase to prostacyclin may contribute to the structural changes and hypertension observed in the pulmonary vasculature of the rat with chronic hypoxia.

The enzyme adenylate cyclase (AC) plays a critical role in regulation of vasodilatation in the developing pulmonary circulation. We characterized the ontogeny of the function of the AC system in the pulmonary vascular smooth muscle (VSM) of fetal lambs during the third trimester. Basal (nonstimulated) AC activity and activity with targeted stimulation of the components of the AC system were determined in pulmonary VSM plasma membranes from fetal lambs at 110-115 days (F-1) and 125-135 days of gestation (F-2) (with term being 144 +/- 3 days) and from pregnant ewes (adult) for comparison. We assessed beta-adrenergic receptor-mediated activity so that we could perform parallel studies of the VSM receptor binding characteristics. Basal AC activity declined 48% from F-1 to F-2 and an additional 13% from F-2 to adult. beta-Adrenergic receptor-stimulated activity was demonstrable only in adult pulmonary VSM membranes even though receptor density and affinity for the agonist were similar in fetal and adult pulmonary VSM. AC activity with NaF stimulation at the level of the G proteins declined 65% from F-1 to F-2 and was similar in F-2 as compared with adult. This indicates that the function of the G proteins or the catalytic subunit of the enzyme decreases from F-1 to F-2. The latter is suggested by the observation that AC activity with direct stimulation of the catalytic subunit with forskolin decreased 45% over this time period.(ABSTRACT TRUNCATED AT 250 WORDS)

alpha 1-Adrenergic receptors mediate vasoconstriction in the pulmonary and systemic vasculature. In sheep the in vivo vasoconstrictor response to alpha 1-adrenergic stimulation is less in the pulmonary circulation compared with the systemic circulation of the fetus, the response increases in both vascular beds with fetal and postnatal development, and it decreases in the systemic vasculature with pregnancy. In an effort to determine the mechanisms underlying these differences, alpha 1-adrenergic receptor binding characteristics were determined by using [3H]prazosin in intrapulmonary and systemic (thoracic aorta) vascular smooth muscle (VSM) from late-gestation fetal lambs and from pregnant and nonpregnant ewes. alpha 1-Adrenergic receptor density was less in fetal intrapulmonary VSM that in fetal aortic VSM (12.4 +/- 1.5 versus 29.4 +/- 3.2 fmol/mg protein, p less than 0.05), and it was less (p less than 0.05) in the fetus compared with the pregnant ewe in both intrapulmonary and aortic VSM (51.0 +/- 5.2 and 76.5 +/- 5.9 fmol/mg protein, respectively). alpha 1-Adrenergic receptor density in intrapulmonary VSM was similar in the pregnant and nonpregnant ewe (61.9 +/- 7.2 fmol/mg protein), whereas in aortic VSM it was less (p less than 0.05) in pregnant ewes compared with nonpregnant ewes (101.0 +/- 5.5 fmol/mg protein). alpha 1-Adrenergic receptor affinity was similar in all the VSM sources tested.(ABSTRACT TRUNCATED AT 250 WORDS)

We tested the hypothesis that simultaneous inhibition of TxA2 synthase and blockade of TxA2/PHG2 receptors is more effective in enhancing thrombolysis and preventing reocclusion after discontinuation of tissue plasminogen activator (t-PA) than either intervention alone. Coronary thrombosis was induced in 35 dogs by placing a copper coil into the left anterior descending coronary artery. Coronary flow was measured with a Doppler flow probe. 30 min after thrombus formation, the animals received saline (controls, n = 10); SQ 29548 (0.4 mg/kg bolus + 0.4 mg/kg per h infusion), a TxA2/PGH2 receptor antagonist (n = 8); dazoxiben (5 mg/kg bolus + 5 mg/kg per h infusion), a TxA2 synthase inhibitor (n = 9); or R 68070 (5 mg/kg bolus + 5 mg/kg per h infusion), a drug that blocks TxA2/PGH2 receptors and inhibits TxA2 synthase (n = 8). Then, all dogs received heparin (200 U/kg) and a bolus of t-PA (80 micrograms/kg) followed by a continuous infusion (8 micrograms/kg per min) for up to 90 min or until reperfusion was achieved. The time to thrombolysis did not change significantly in SQ 29548-treated dogs as compared with controls (42 +/- 5 vs. 56 +/- 7 min, respectively, P = NS), but it was significantly shortened by R 68070 and dazoxiben (11 +/- 2 and 25 +/- 6 min, respectively, P less than 0.001 vs. controls and SQ 29548-treated dogs). R 68070 administration resulted in a lysis time significantly shorter than that observed in the dazoxiben-treated group (P less than 0.01). Reocclusion was observed in eight of eight control dogs, five of seven SQ 29548-treated dogs, seven of nine dazoxiben-treated dogs, and zero of eight R 68070-treated animals (P less than 0.001). TxB2 and 6-keto-PGF1 alpha, measured in blood samples obtained from the coronary artery distal to the thrombus, were significantly increased at reperfusion and at reocclusion in control animals and in dogs receiving SQ 29548. R 68070 and dazoxiben prevented the increase in plasma TxB2 levels, whereas 6-keto-PGF1 alpha levels were significantly increased with respect to control and SQ 29548-treated dogs. Thus, simultaneous inhibition of TxA2 synthase and blockade of TxA2/PGH2 receptors is more effective than either intervention alone in this experimental model in enhancing thrombolysis and preventing reocclusion after t-PA administration.

Oxygen-derived free radicals have been implicated in damage to membrane phospholipids leading to alterations in membrane function. The purpose of this study was to investigate alterations in intracellular ionic calcium (Ca2+) levels and Ca2+ transients, cellular morphology, conjugated diene levels, arachidonate release, and lactate dehydrogenase release resulting from the exposure of cultured neonatal rat ventricular myocytes to a xanthine oxidase catalyzed free radical generating system capable of producing superoxide and hydroxyl radicals. The ability of alpha-tocopherol to prevent alterations due to free radical exposure was investigated. For measurements of Ca2+, myocytes grown on coverslips for 3-4 days were loaded with fura-2/AM and studied by microspectrofluorometry. Control myocytes superfused with a physiological buffer or buffer containing purine and iron-loaded transferrin exhibited Ca2+ transients associated with spontaneous contractions. For control, buffer perfused myocytes (n = 4), the fura-2 340/380 ratios were 0.5 +/- 0.1 (mean +/- S.E.) and 1.6 +/- 0.03 at the minimum and maximum, respectively, of the Ca2+ transient, after 1 h of perfusion. Exposure to the free radical generating solution (n = 14) altered intracellular Ca2+. The 340/380 minimum ratio was 639% of the control value after approximately 30-70 mins with cessation of normal Ca2+ transients. Bleb development was associated with increased Ca2+. Myocytes reperfused with control medium continued to exhibit an elevated minimum fura-2 ratio at 687% of control. Myocytes pretreated with 10 microM alpha-tocopherol (n = 13) for 18-24 h and exposed to free radicals did not exhibit increases in intracellular Ca2+, having a minimum 340/380 ratio of 0.5 +/- 0.1 after 60-90 mins, and although myocytes often ceased contracting, they resumed spontaneous Ca2+ transients with control medium reperfusion and also maintained normal structure. Exposure of myocyte cultures to free radical generating solutions resulted in increased levels of conjugated dienes and increased release of [3H]arachidonate and lactate dehydrogenase compared to control values after 1 h. alpha-Tocopherol treatment attenuated the increase in conjugated diene levels, and the release of [3H]arachidonate and lactate dehydrogenase. Thus, free radicals alter intracellular Ca2+, conjugated dienes and membrane structure indicating their ability to induce altered ionic homeostasis in association with myocardial membrane damage. alpha-Tocopherol decreased free radical mediated injury.

Prolonged depression of segmental systolic thickening after brief coronary artery occlusion may result principally from events during reperfusion rather than during the ischemic interval. Thus, cellular calcium overload at reperfusion may be a mediator of contractile dysfunction after brief ischemia, and reduction of calcium entry by diltiazem, a calcium channel antagonist, may enhance recovery of systolic thickening after brief periods of ischemia. Thirteen awake unsedated dogs instrumented with hemodynamic catheters, left anterior descending coronary artery occluders and five to six pairs of intramyocardial sonomicrometers underwent two 15 min coronary artery occlusions with 24 h reperfusion. The order of infusion of diltiazem (15 micrograms/kg per min) or saline solution was alternated. Systolic thickening, hemodynamic variables and regional myocardial blood flow were measured serially over 24 h. Despite equally severe ischemic dysfunction during coronary occlusion, diltiazem-treated segments with systolic thinning during ischemia recovered control segmental thickening significantly earlier than saline solution-treated segments (at 30 versus 180 min of reperfusion). Blood pressure was mildly decreased during diltiazem treatment; therefore, a second group of 10 dogs underwent a similar occlusion and reflow period during infusion of nitroprusside to lower mean arterial pressure equivalently. Decreases in blood pressure in this group resulted in some improvement in segmental systolic function; however, this did not reach statistical significance at any time. Regional myocardial blood flows were similar in the saline solution- and diltiazem-treated groups during ischemia and reflow. Thus, it is concluded that 1) diltiazem infusion significantly enhanced recovery of segmental systolic thickening after 15 min of ischemia and 24 h of reperfusion; 2) the enhancement in segmental systolic function could not entirely be attributed to decreased mean arterial pressure; 3) improvement in postischemic segmental ventricular function was seen only in those segments with systolic thinning during ischemia; thus, segments with the most severe ischemic dysfunction benefited most; and 4) there were no important differences in regional myocardial blood flow during ischemia and reperfusion between saline- and diltiazem-treated animals.

Defective expression of low density lipoprotein (LDL) receptors is the basic genetic defect in human familial hypercholesterolaemia (FH) and its animal counterpart, the Watanabe heritable hyperlipidaemic (WHHL) rabbit. The pathologic manifestations of human FH were evaluated based on the study of material from six subjects with homozygous FH and a review of the literature. This analysis indicated that homozygous FH is characterized by accelerated atherosclerosis and prominent lipid accumulation in macrophages and other stromal cells of the aortic and mitral valves, skin, tendon, and, variably, in other extravascular sites. Disease progression was studied in the WHHL rabbit. From birth to 1 year, WHHL rabbits show evidence of progressive disease of the aorta with accumulation of birefringent lipid in intimal lesions, including fatty streaks, raised foam cell lesions, and plaques (atheromas), as well as in the media. At 1-2 years, WHHL rabbits develop coronary atherosclerosis and focal extravascular lipid deposits, including subcutaneous and tendinous xanthomas. Vascular lesion development is associated with adhesion of monocytes and other leucocytes to the endothelium. The cells of the intimal lesions are lipid-containing macrophages, smooth muscle cells and foam cells. Most of the intracellular lipid is in the form of non-membrane-bound neutral lipid droplets indicating that the cytoplasm is the major site of lipid storage. Similar ultrastructural features are shown by human FH lesions. Observations are reviewed regarding therapeutic approaches aimed at altering the pathologic expression of familial hypercholesterolaemia, including the negative results of treatment of WHHL rabbits with the calcium-channel antagonist, verapamil, and omega-3 fatty acids.

A computational model was recently designed to simulate cellular changes in the T cell immune system. The model was validated by simulating cell changes in viral infections which target the same CD4+ T cell, yet cause either hyperplastic, aplastic or neoplastic responses. Respective case material for comparison was available from human infections with human herpesvirus-6 (HHV-6), human immunodeficiency virus (HIV-1) or human T cell leukemia virus (HTLV-1). Starting with cell values for a healthy human individual, factorial changes that influence the individual course of the various infections were determined by an algorithm search procedure. Such factorial differences determining a clinical course with aplasia, hyperplasia or neoplasia are outlined and further discussed in this paper.

We describe a biodynamical regulatory model of the human T-cell system based on a set of coupled ordinary differential equations. The model incorporates subsystems representing bone marrow, thymus and peripheral (mature T-cell) compartments and allows simulations of viral challenges to the system which lead to various disorders such as leukemia-like syndromes, AIDS, and herpes infections. We provide two examples, one of normative data across the lifespan and the other acute human herpes virus (HHV-6) infection.