Donatas Kraskauskas

Chronic Pulmonary Artery Pressure Elevation Is Insufficient to Explain Right Heart Failure

Background— The most important determinant of longevity in pulmonary arterial hypertension is right ventricular (RV) function, but in contrast to experimental work elucidating the pathobiology of left ventricular failure, there is a paucity of data on the cellular and molecular mechanisms of RV failure. Methods and Results— A mechanical animal model of chronic progressive RV pressure overload (pulmonary artery banding, not associated with structural alterations of the lung circulation) was compared with an established model of angioproliferative pulmonary hypertension associated with fatal RV failure. Isolated RV pressure overload induced RV hypertrophy without failure, whereas in the context of angioproliferative pulmonary hypertension, RV failure developed that was associated with myocardial apoptosis, fibrosis, a decreased RV capillary density, and a decreased vascular endothelial growth factor mRNA and protein expression despite increased nuclear stabilization of hypoxia-induced factor-1α. Induction of myocardial nuclear factor E2-related factor 2 and heme-oxygenase 1 with a dietary supplement (Protandim) prevented fibrosis and capillary loss and preserved RV function despite continuing pressure overload. Conclusions— These data brought into question the commonly held concept that RV failure associated with pulmonary hypertension is due strictly to the increased RV afterload.

Rho Kinase–Mediated Vasoconstriction Is Important in Severe Occlusive Pulmonary Arterial Hypertension in Rats

Vascular remodeling, rather than vasoconstriction, is believed to account for high vascular resistance in severe pulmonary arterial hypertension (PAH). We have found previously that acute Rho kinase inhibition nearly normalizes PAH in chronically hypoxic rats that have no occlusive neointimal lesions. Here we examined whether Rho kinase-mediated vasoconstriction was also important in a rat model of severe occlusive PAH. Adult rats were exposed to chronic hypoxia (≈10% O2) after subcutaneous injection of the vascular endothelial growth factor receptor inhibitor SUGEN 5416. Hemodynamic measurements were made in anesthetized rats after 2 weeks of hypoxia (early group) and 3 weeks of hypoxia plus 2 weeks of normoxia (late group). Both groups developed PAH, with greater severity in the late group. In the early group, intravenous fasudil was more effective than intravenous bradykinin, inhaled NO, or intravenous iloprost in reducing right ventricular systolic pressure. Despite more occlusive vascular lesions, fasudil also markedly reduced right ventricular systolic pressure in late-stage rats. Blood-perfused lungs from late-stage rats showed spontaneous vasoconstriction, which was reversed partially by the endothelin A receptor blocker BQ123 and completely by fasudil or Y-27632. Phosphorylation of MYPT1, a downstream target of Rho kinase, was increased in lungs from both groups of rats, and fasudil (intravenous) reversed the increased phosphorylation in the late group. Thus, in addition to structural occlusion, Rho kinase-mediated vasoconstriction is an important component of severe PAH in SUGEN 5416/hypoxia-exposed rats, and PAH can be significantly reduced in the setting of a severely remodeled lung circulation if an unconventional vasodilator is used.

Adrenergic Receptor Blockade Reverses Right Heart Remodeling and Dysfunction in Pulmonary Hypertensive Rats

RATIONALE Most patients with pulmonary arterial hypertension (PAH) die from right heart failure. Beta-adrenergic receptor blockade reduces mortality by about 30% in patients with left-sided systolic heart failure, but is not used in PAH. OBJECTIVES To assess the effect of the adrenergic receptor blocker carvedilol on the pulmonary circulation and right heart in experimental pulmonary hypertension in rats. METHODS Angioproliferative pulmonary hypertension was induced in rats by combined exposure to the vascular endothelial growth factor-receptor antagonist SU5416 and hypoxia. Carvedilol treatment was started after establishment of pulmonary hypertension and right heart dysfunction. MEASUREMENTS AND MAIN RESULTS Compared with vehicle-treated animals, treatment with carvedilol resulted in increased exercise endurance; improved right ventricular (RV) function (increased tricuspid annular plane systolic excursion and decreased RV dilatation); and an increased cardiac output. The morphology of the pulmonary vessels and the RV afterload were not affected by carvedilol. Carvedilol treatment was associated with enhancement of RV fetal gene reactivation, increased protein kinase G (PKG) activity, and a reduction in capillary rarefaction and fibrosis. Metoprolol had similar but less pronounced effects in the SU5416 and hypoxia model. Cardioprotective effects were noted of both carvedilol and metoprolol in the monocrotaline model. In the case of carvedilol, but not metoprolol, part of these effects resulted from a prevention of monocrotaline-induced lung remodeling. CONCLUSIONS Adrenergic receptor blockade reverses RV remodeling and improves RV function in experimental pulmonary hypertension. Beta-adrenergic receptor blockers are not recommended in humans with PAH before their safety and efficacy are assessed in well-designed clinical trials.

The monocrotaline model of pulmonary hypertension in perspective

Severe forms of pulmonary arterial hypertension (PAH) are characterized by various degrees of remodeling of the pulmonary arterial vessels, which increases the pulmonary vascular resistance and right ventricular afterload, thus contributing to the development of right ventricle dysfunction and failure. Recent years have seen advances in the understanding of the pathobiology of PAH; however, many important questions remain unanswered. Elucidating the pathobiology of PAH continues to be critical to design new effective therapeutic strategies, and appropriate animal models of PAH are necessary to achieve the task. Although the monocrotaline rat model of PAH has contributed to a better understanding of vascular remodeling in pulmonary hypertension, we question the validity of this model as a preclinically relevant model of severe plexogenic PAH. Here we review pertinent publications that either have been forgotten or ignored, and we reexamine the monocrotaline model in the context of human forms of PAH.

Suppression of histone deacetylases worsens right ventricular dysfunction after pulmonary artery banding in rats.

RATIONALE Inhibitors of histone deacetylases (HDACs) reduce pressure-overload-induced left ventricular hypertrophy and dysfunction, but their effects on right ventricular (RV) adaptation to pressure overload are unknown. OBJECTIVES Determine the effect of the broad-spectrum HDAC inhibitors trichostatin A (TSA) and valproic acid (VPA) on RV function and remodeling after pulmonary artery banding (PAB) in rats. METHODS Chronic progressive RV pressure-overload was induced in rats by PAB. After establishment of adaptive RV hypertrophy 4 weeks after surgery, rats were treated for 2 weeks with vehicle, TSA, or VPA. RV function and remodeling were determined using echocardiography, invasive hemodynamic measurements, immunohistochemistry, and molecular analyses after 2 weeks of HDAC inhibition. The effects of TSA were determined on the expression of proangiogenic and prohypertrophic genes in human myocardial fibroblasts and microvascular endothelial cells. MEASUREMENTS AND MAIN RESULTS TSA treatment did not prevent the development of RV hypertrophy and was associated with RV dysfunction, capillary rarefaction, fibrosis, and increased rates of myocardial cell death. Similar results were obtained with the structurally unrelated HDAC inhibitor VPA. With TSA treatment, a reduction was found in expression of vascular endothelial growth factor and angiopoietin-1, which proteins are involved in vascular adaptation to pressure-overload. TSA dose-dependently suppressed vascular endothelial growth factor, endothelial nitric oxide synthase, and angiopoietin-1 expression in cultured myocardial endothelial cells, which effects were mimicked by selective gene silencing of several class I and II HDACs. CONCLUSIONS HDAC inhibition is associated with dysfunction and worsened remodeling of the pressure-overloaded RV. The detrimental effects of HDAC inhibition on the pressure-overloaded RV may come about via antiangiogenic or proapoptotic effects.

A brief overview of mouse models of pulmonary arterial hypertension: problems and prospects

Many chronic pulmonary diseases are associated with pulmonary hypertension (PH) and pulmonary vascular remodeling, which is a term that continues to be used to describe a wide spectrum of vascular abnormalities. Pulmonary vascular structural changes frequently increase pulmonary vascular resistance, causing PH and right heart failure. Although rat models had been standard models of PH research, in more recent years the availability of genetically engineered mice has made this species attractive for many investigators. Here we review a large amount of data derived from experimental PH reports published since 1996. These studies using wild-type and genetically designed mice illustrate the challenges and opportunities provided by these models. Hemodynamic measurements are difficult to obtain in mice, and right heart failure has not been investigated in mice. Anatomical, cellular, and genetic differences distinguish mice and rats, and pharmacogenomics may explain the degree of PH and the particular mode of pulmonary vascular adaptation and also the response of the right ventricle.

p53 Gene deficiency promotes hypoxia-induced pulmonary hypertension and vascular remodeling in mice

Chronic hypoxia induces pulmonary arterial remodeling, resulting in pulmonary hypertension and right ventricular hypertrophy. Hypoxia has been implicated as a physiological stimulus for p53 induction and hypoxia-inducible factor-1α (HIF-1α). However, the subcellular interactions between hypoxic exposure and expression of p53 and HIF-1α remain unclear. To examine the role of p53 and HIF-1α expression on hypoxia-induced pulmonary arterial remodeling, wild-type (WT) and p53 knockout (p53KO) mice were exposed to either normoxia or hypoxia for 8 wk. Following chronic hypoxia, both genotypes demonstrated elevated right ventricular pressures, right ventricular hypertrophy as measured by the ratio of the right ventricle to the left ventricle plus septum weights, and vascular remodeling. However, the right ventricular systolic pressures, the ratio of the right ventricle to the left ventricle plus septum weights, and the medial wall thickness of small vessels were significantly greater in the p53KO mice than in the WT mice. The p53KO mice had lower levels of p21 and miR34a expression, and higher levels of HIF-1α, VEGF, and PDGF expression than WT mice following chronic hypoxic exposure. This was associated with a higher proliferating cell nuclear antigen expression of pulmonary artery in p53KO mice. We conclude that p53 plays a critical role in the mitigation of hypoxia-induced small pulmonary arterial remodeling. By interacting with p21 and HIF-1α, p53 may suppress hypoxic pulmonary arterial remodeling and pulmonary arterial smooth muscle cell proliferation under hypoxia.

Metabolic Gene Remodeling and Mitochondrial Dysfunction in Failing Right Ventricular Hypertrophy Secondary to Pulmonary Arterial Hypertension

Background— Right ventricular (RV) dysfunction (RVD) is the most frequent cause of death in patients with pulmonary arterial hypertension. Although abnormal energy substrate use has been implicated in the development of chronic left heart failure, data describing such metabolic remodeling in RVD remain incomplete. Thus, we sought to characterize metabolic gene expression changes and mitochondrial dysfunction in functional and dysfunctional RV hypertrophy. Methods and Results— Two different rat models of RV hypertrophy were studied. The model of RVD (SU5416/hypoxia) exhibited a significantly decreased gene expression of peroxisome proliferator-activated receptor-&ggr; coactivator-1&agr;, peroxisome proliferator-activated receptor-&agr; and estrogen-related receptor-&agr;. The expression of multiple peroxisome proliferator-activated receptor-&ggr; coactivator-1&agr; target genes required for fatty acid oxidation was similarly decreased. Decreased peroxisome proliferator-activated receptor-&ggr; coactivator-1&agr; expression was also associated with a net loss of mitochondrial protein and oxidative capacity. Reduced mitochondrial number was associated with a downregulation of transcription factor A, mitochondrial, and other genes required for mitochondrial biogenesis. Electron microscopy demonstrated that, in RVD tissue, mitochondria had abnormal shape and size. Lastly, respirometric analysis demonstrated that mitochondria isolated from RVD tissue had a significantly reduced ADP-stimulated (state 3) rate for complex I. Conversely, functional RV hypertrophy in the pulmonary artery banding model showed normal expression of peroxisome proliferator-activated receptor-&ggr; coactivator-1&agr;, whereas the expression of fatty acid oxidation genes was either preserved or unregulated. Moreover, pulmonary artery banding-RV tissue exhibited preserved transcription factor A mitochondrial expression and mitochondrial respiration despite elevated RV pressure-overload. Conclusions— Right ventricular dysfunction, but not functional RV hypertrophy in rats, demonstrates a gene expression profile compatible with a multilevel impairment of fatty acid metabolism and significant mitochondrial dysfunction, partially independent of chronic pressure-overload.

Mechanisms of attenuation of abdominal sepsis induced acute lung injury by ascorbic acid.

Bacterial infections of the lungs and abdomen are among the most common causes of sepsis. Abdominal peritonitis often results in acute lung injury (ALI). Recent reports demonstrate a potential benefit of parenteral vitamin C [ascorbic acid (AscA)] in the pathogenesis of sepsis. Therefore we examined the mechanisms of vitamin C supplementation in the setting of abdominal peritonitis-mediated ALI. We hypothesized that vitamin C supplementation would protect lungs by restoring alveolar epithelial barrier integrity and preventing sepsis-associated coagulopathy. Male C57BL/6 mice were intraperitoneally injected with a fecal stem solution to induce abdominal peritonitis (FIP) 30 min prior to receiving either AscA (200 mg/kg) or dehydroascorbic acid (200 mg/kg). Variables examined included survival, extent of ALI, pulmonary inflammatory markers (myeloperoxidase, chemokines), bronchoalveolar epithelial permeability, alveolar fluid clearance, epithelial ion channel, and pump expression (aquaporin 5, cystic fibrosis transmembrane conductance regulator, epithelial sodium channel, and Na(+)-K(+)-ATPase), tight junction protein expression (claudins, occludins, zona occludens), cytoskeletal rearrangements (F-actin polymerization), and coagulation parameters (thromboelastography, pro- and anticoagulants, fibrinolysis mediators) of septic blood. FIP-mediated ALI was characterized by compromised lung epithelial permeability, reduced alveolar fluid clearance, pulmonary inflammation and neutrophil sequestration, coagulation abnormalities, and increased mortality. Parenteral vitamin C infusion protected mice from the deleterious consequences of sepsis by multiple mechanisms, including attenuation of the proinflammatory response, enhancement of epithelial barrier function, increasing alveolar fluid clearance, and prevention of sepsis-associated coagulation abnormalities. Parenteral vitamin C may potentially have a role in the management of sepsis and ALI associated with sepsis.

MicroRNA-199a-5p Is Associated With Hypoxia-Inducible Factor-1α Expression in Lungs From Patients With COPD

BACKGROUND MicroRNAs (miRNAs) are small noncoding RNAs that silence target gene expression posttranscriptionally, and their impact on gene expression has been reported in various diseases. It has been reported that the expression of the hypoxia-inducible factor-1α (HIF-1α) is reduced and that of p53 is increased in lungs from patients with COPD. However, the role of miRNAs associated with these genes in lungs from patients with COPD is unknown. METHODS Lung tissue samples from 55 patients were included in this study. Total RNA, miRNA, and protein were extracted from lung tissues and used for reverse transcriptase polymerase chain reaction and Western blot analysis. Cell culture experiments were performed using cultured human pulmonary microvascular endothelial cells (HPMVECs). RESULTS miR-34a and miR-199a-5p expressions were increased, and the phosphorylation of AKT was decreased in the lung tissue samples of patients with COPD. The miR-199a-5p expression was correlated with HIF-1α protein expression in the lungs of patients with COPD. Transfection of HPMVECs with the miR-199a-5p precursor gene decreased HIF-1α protein expression, and transfection with the miR-34a precursor gene increased miR-199a-5p expression. CONCLUSIONS These data suggest that miR-34a and miR-199a-5p contribute to the pathogenesis of COPD, and these miRNAs may also affect the HIF-1α-dependent lung structure maintenance program.