Sachindra Raj Joshi

Lipid nanoparticle delivery of a microRNA-145 inhibitor improves experimental pulmonary hypertension

Therapies that exploit RNA interference (RNAi) hold great potential for improving disease outcomes. However, there are several challenges that limit the application of RNAi therapeutics. One of the most important challenges is effective delivery of oligonucleotides to target cells and reduced delivery to non-target cells. We have previously developed a functionalized cationic lipopolyamine (Star:Star-mPEG-550) for in vivo delivery of siRNA to pulmonary vascular cells. This optimized lipid formulation enhances the retention of siRNA in mouse lungs and achieves significant knockdown of target gene expression for at least 10days following a single intravenous injection. Although this suggests great potential for developing lung-directed RNAi-based therapies, the application of Star:Star-mPEG mediated delivery of RNAi based therapies for pulmonary vascular diseases such as pulmonary arterial hypertension (PAH) remains unknown. We identified differential expression of several microRNAs known to regulate cell proliferation, cell survival and cell fate that are associated with development of PAH, including increased expression of microRNA-145 (miR-145). Here we test the hypothesis that Star:Star-mPEG mediated delivery of an antisense oligonucleotide against miR-145 (antimiR-145) will improve established PAH in rats. We performed a series of experiments testing the in vivo distribution, toxicity, and efficacy of Star:Star-mPEG mediated delivery of antimiR-145 in rats with Sugen-5416/hypoxia induced PAH. We showed that after subchronic therapy of three intravenous injections over 5weeks at 2mg/kg, antimiR-145 accumulated in rat lung tissue and reduced expression of endogenous miR-145. Using a novel in situ hybridization approach, we demonstrated substantial distribution of antimiR-145 in the lungs as well as the liver, kidney, and spleen. We assessed toxic effects of Star:Star-mPEG/antimiR-145 with serial complete blood counts of leukocytes and serum metabolic panels, gross pathology, and histopathology and did not detect significant off-target effects. AntimiR-145 reduced the degree of pulmonary arteriopathy, reduced the severity of pulmonary hypertension, and reduced the degree of cardiac dysfunction. The results establish effective and low toxicity of lung delivery of a miRNA-145 inhibitor using functionalized cationic lipopolyamine nanoparticles to repair pulmonary arteriopathy and improve cardiac function in rats with severe PAH.

MicroRNAs-control of essential genes: Implications for pulmonary vascular disease

During normal lung development and in lung diseases structural cells in the lungs adapt to permit changes in lung function. Fibroblasts, myofibroblasts, smooth muscle, epithelial cells, and various progenitor cells can all undergo phenotypic modulation. In the pulmonary vasculature occlusive vascular lesions that occur in severe pulmonary arterial hypertension are multifocal, polyclonal lesions containing cells presumed to have undergone phenotypic transition resulting in altered proliferation, cell lifespan or contractility. Dynamic changes in gene expression and protein composition that underlie processes responsible for such cellular plasticity are not fully defined. Advances in molecular biology have shown that multiple classes of ribonucleic acid (RNA) collaborate to establish the set of proteins expressed in a cell. Both coding Messenger Ribonucleic acid (mRNA) and small noncoding RNAs (miRNA) act via multiple parallel signaling pathways to regulate transcription, mRNA processing, mRNA stability, translation and possibly protein lifespan. Rapid progress has been made in describing dynamic features of miRNA expression and miRNA function in some vascular tissues. However posttranscriptional gene silencing by microRNA-mediated mRNA degradation and translational blockade is not as well defined in the pulmonary vasculature. Recent progress in defining miRNAs that modulate vascular cell phenotypes is reviewed to illustrate both functional and therapeutic significance of small noncoding RNAs in pulmonary arterial hypertension.

MicroRNA-140 is elevated and mitofusin-1 is downregulated in the right ventricle of the Sugen5416/hypoxia/normoxia model of pulmonary arterial hypertension.

Heart failure, a major cause of morbidity and mortality in patients with pulmonary arterial hypertension (PAH), is an outcome of complex biochemical processes. In this study, we determined changes in microRNAs (miRs) in the right and left ventricles of normal and PAH rats. Using an unbiased quantitative miR microarray analysis, we found 1) miR-21-5p, miR-31-5 and 3p, miR-140-5 and 3p, miR-208b-3p, miR-221-3p, miR-222-3p, miR-702-3p, and miR-1298 were upregulated (>2-fold; P < 0.05) in the right ventricle (RV) of PAH compared with normal rats; 2) miR-31-5 and 3p, and miR-208b-3p were upregulated (>2-fold; P < 0.05) in the left ventricle plus septum (LV+S) of PAH compared with normal rats; 3) miR-187-5p, miR-208a-3p, and miR-877 were downregulated (>2-fold; P < 0.05) in the RV of PAH compared with normal rats; and 4) no miRs were up- or downregulated with >2-fold in LV+S compared with RV of PAH and normal. Upregulation of miR-140 and miR-31 in the hypertrophic RV was further confirmed by quantitative PCR. Interestingly, compared with control rats, expression of mitofusin-1 (MFN1), a mitochondrial fusion protein that regulates apoptosis, and which is a direct target of miR-140, was reduced in the RV relative to LV+S of PAH rats. We found a correlation between increased miR-140 and decreased MFN1 expression in the hypertrophic RV. Our results also demonstrated that upregulation of miR-140 and downregulation of MFN1 correlated with increased RV systolic pressure and hypertrophy. These results suggest that miR-140 and MFN1 play a role in the pathogenesis of PAH-associated RV dysfunction.

Glucose-6-phosphate dehydrogenase plays a critical role in hypoxia-induced CD133+ progenitor cells self-renewal and stimulates their accumulation in the lungs of pulmonary hypertensive rats

Although hypoxia is detrimental to most cell types, it aids survival of progenitor cells and is associated with diseases like cancer and pulmonary hypertension in humans. Therefore, understanding the underlying mechanisms that promote survival of progenitor cells in hypoxia and then developing novel therapies to stop their growth in hypoxia-associated human diseases is important. Here we demonstrate that the proliferation and growth of human CD133(+) progenitor cells, which contribute to tumorigenesis and the development of pulmonary hypertension, are increased when cultured under hypoxic conditions. Furthermore, glucose-6-phosphate dehydrogenase (G6PD) activity was increased threefold in hypoxic CD133(+) cells. The increased G6PD activity was required for CD133(+) cell proliferation, and their growth was arrested by G6PD inhibition or knockdown. G6PD activity upregulated expression of HIF1α, cyclin A, and phospho-histone H3, thereby promoting CD133(+) cell dedifferentiation and self-renewal and altering cell cycle regulation. When CD133(+) cells were cocultured across a porous membrane from pulmonary artery smooth muscle cells (PASMCs), G6PD-dependent H2O2 production and release by PASMCs recruited CD133(+) cells to the membrane, where they attached and expressed smooth muscle markers (α-actin and SM22α). Inhibition of G6PD reduced smooth muscle marker expression in CD133(+) cells under normoxia but not hypoxia. In vivo, CD133(+) cells colocalized with G6PD(+) cells in the perivascular region of lungs from rats with hypoxia-induced pulmonary hypertension. Finally, inhibition of G6PD by dehydroepiandrosterone in pulmonary arterial hypertensive rats nearly abolished CD133(+) cell accumulation around pulmonary arteries and the formation of occlusive lesions. These observations suggest G6PD plays a key role in increasing hypoxia-induced CD133(+) cell survival in hypertensive lungs that differentiate to smooth muscle cells and contribute to pulmonary arterial remodeling during development of pulmonary hypertension.

20-HETE-induced mitochondrial superoxide production and inflammatory phenotype in vascular smooth muscle is prevented by glucose-6-phosphate dehydrogenase inhibition

20-Hydroxyeicosatetraeonic acid (20-HETE) produced by cytochrome P-450 monooxygenases in NADPH-dependent manner is proinflammatory, and it contributes to the pathogenesis of systemic and pulmonary hypertension. In this study, we tested the hypothesis that inhibition of glucose-6-phosphate dehydrogenase (G6PD), a major source of NADPH in the cell, prevents 20-HETE synthesis and 20-HETE-induced proinflammatory signaling that promotes secretory phenotype of vascular smooth muscle cells. Lipidomic analysis indicated that G6PD inhibition and knockdown decreased 20-HETE levels in pulmonary arteries as well as 20-HETE-induced 1) mitochondrial superoxide production, 2) activation of mitogen-activated protein kinase 1 and 3, 3) phosphorylation of ETS domain-containing protein Elk-1 that activate transcription of tumor necrosis factor-α gene (Tnfa), and 4) expression of tumor necrosis factor-α (TNF-α). Moreover, inhibition of G6PD increased protein kinase G1α activity, which, at least partially, mitigated superoxide production and Elk-1 and TNF-α expression. Additionally, we report here for the first time that 20-HETE repressed miR-143, which suppresses Elk-1 expression, and miR-133a, which is known to suppress synthetic/secretory phenotype of vascular smooth muscle cells. In summary, our findings indicate that 20-HETE elicited mitochondrial superoxide production and promoted secretory phenotype of vascular smooth muscle cells by activating MAPK1-Elk-1, all of which are blocked by inhibition of G6PD.

Sphingosine-1-phosphate is involved in the occlusive arteriopathy of pulmonary arterial hypertension

Despite several advances in the pathobiology of pulmonary arterial hypertension (PAH), its pathogenesis is not completely understood. Current therapy improves symptoms but has disappointing effects on survival. Sphingosine-1-phosphate (S1P) is a lysophospholipid synthesized by sphingosine kinase 1 (SphK1) and SphK2. Considering the regulatory roles of S1P in several tissues leading to vasoconstriction, inflammation, proliferation, and fibrosis, we investigated whether S1P plays a role in the pathogenesis of PAH. To test this hypothesis, we used plasma samples and lung tissue from patients with idiopathic PAH (IPAH) and the Sugen5416/hypoxia/normoxia rat model of occlusive PAH. Our study revealed an increase in the plasma concentration of S1P in patients with IPAH and in early and late stages of PAH in rats. We observed increased expression of both SphK1 and SphK2 in the remodeled pulmonary arteries of patients with IPAH and PAH rats. Exogenous S1P stimulated the proliferation of cultured rat pulmonary arterial endothelial and smooth-muscle cells. We also found that 3 weeks of treatment of late-stage PAH rats with an SphK1 inhibitor reduced the increased plasma levels of S1P and the occlusive pulmonary arteriopathy. Although inhibition of SphK1 improved cardiac index and the total pulmonary artery resistance index, it did not reduce right ventricular systolic pressure or right ventricular hypertrophy. Our study supports that S1P is involved in the pathogenesis of occlusive arteriopathy in PAH and provides further evidence that S1P signaling may be a novel therapeutic target.

Involvement of gap junctions between smooth muscle cells in sustained hypoxic pulmonary vasoconstriction development: a potential role for 15-HETE and 20-HETE.

In response to hypoxia, the pulmonary artery normally constricts to maintain optimal ventilation-perfusion matching in the lung, but chronic hypoxia leads to the development of pulmonary hypertension. The mechanisms of sustained hypoxic pulmonary vasoconstriction (HPV) remain unclear. The aim of this study was to determine the role of gap junctions (GJs) between smooth muscle cells (SMCs) in the sustained HPV development and involvement of arachidonic acid (AA) metabolites in GJ-mediated signaling. Vascular tone was measured in bovine intrapulmonary arteries (BIPAs) using isometric force measurement technique. Expression of contractile proteins was determined by Western blot. AA metabolites in the bath fluid were analyzed by mass spectrometry. Prolonged hypoxia elicited endothelium-independent sustained HPV in BIPAs. Inhibition of GJs by 18β-glycyrrhetinic acid (18β-GA) and heptanol, nonspecific blockers, and Gap-27, a specific blocker, decreased HPV in deendothelized BIPAs. The sustained HPV was not dependent on Ca(2+) entry but decreased by removal of Ca(2+) and by Rho-kinase inhibition with Y-27632. Furthermore, inhibition of GJs decreased smooth muscle myosin heavy chain (SM-MHC) expression and myosin light chain phosphorylation in BIPAs. Interestingly, inhibition of 15- and 20-hydroxyeicosatetraenoic acid (HETE) synthesis decreased HPV in deendothelized BIPAs. 15-HETE- and 20-HETE-stimulated constriction of BIPAs was inhibited by 18β-GA and Gap-27. Application of 15-HETE and 20-HETE to BIPAs increased SM-MHC expression, which was also suppressed by 18β-GA and by inhibitors of lipoxygenase and cytochrome P450 monooxygenases. More interestingly, 15,20-dihydroxyeicosatetraenoic acid and 20-OH-prostaglandin E2, novel derivatives of 20-HETE, were detected in tissue bath fluid and synthesis of these derivatives was almost completely abolished by 18β-GA. Taken together, our novel findings show that GJs between SMCs are involved in the sustained HPV in BIPAs, and 15-HETE and 20-HETE, through GJs, appear to mediate SM-MHC expression and contribute to the sustained HPV development.

Cyp2c44 gene disruption exacerbated pulmonary hypertension and heart failure in female but not male mice

Epoxyeicosatrienoicacids (EETs), synthesized from arachidonic acid by epoxygenases of the CYP2C and CYP2J gene subfamilies, contribute to hypoxic pulmonary vasoconstriction (HPV) in mice. Despite their roles in HPV, it is controversial whether EETs mediate or ameliorate pulmonary hypertension (PH). A recent study showed that deficiency of Cyp2j did not protect male and female mice from hypoxia-induced PH. Since CYP2C44 is a functionally important epoxygenase, we hypothesized that knockout of the Cyp2c44 gene would protect both sexes of mice from hypoxia-induced PH. We tested this hypothesis in wild-type (WT) and Cyp2c44 knockout (Cyp2c44−/−) mice exposed to normoxia (room air) and hypoxia (10% O2) for 5 weeks. Exposure of WT and Cyp2c44−/− mice to hypoxia resulted in pulmonary vascular remodeling, increased pulmonary artery resistance, and decreased cardiac function in both sexes. However, in female Cyp2c44−/− mice, compared with WT mice, (1) pulmonary artery resistance and right ventricular hypertrophy were greater, (2) cardiac index was lower, (3) left ventricular and arterial stiffness were higher, and (4) plasma aldosterone levels were higher, but (5) there was no difference in levels of EET in lungs and heart. Paradoxically and unexpectedly, we found that Cyp2c44 disruption exacerbated hypoxia-induced PH in female but not male mice. We attribute exacerbated PH in female Cyp2c44−/− mice to elevated aldosterone and as-yet-unknown systemic factors. Therefore, we suggest a role for the human CYP2C genes in protecting women from severe PH and that this could be one of the underlying causes for a better 5-year survival rate in women than in men.

Elevated 20-HETE in metabolic syndrome regulates arterial stiffness and systolic hypertension via MMP12 activation

Arterial stiffness plays a causal role in development of systolic hypertension. 20-hydroxyeicosatetraeonic acid (20-HETE), a cytochrome P450 (CYP450)-derived arachidonic acid metabolite, is known to be elevated in resistance arteries in hypertensive animal models and loosely associated with obesity in humans. However, the role of 20-HETE in the regulation of large artery remodeling in metabolic syndrome has not been investigated. We hypothesized that elevated 20-HETE in metabolic syndrome increases matrix metalloproteinase 12 (MMP12) activation leading to increased degradation of elastin, increased large artery stiffness and increased systolic blood pressure. 20-HETE production was increased ~7 fold in large, conduit arteries of metabolic syndrome (JCR:LA-cp, JCR) vs. normal Sprague-Dawley (SD) rats. This correlated with increased elastin degradation (~7 fold) and decreased arterial compliance (~75% JCR vs. SD). 20-HETE antagonists blocked elastin degradation in JCR rats concomitant with blocking MMP12 activation. 20-HETE antagonists normalized, and MMP12 inhibition (pharmacological and MMP12-shRNA-Lnv) significantly improved (~50% vs. untreated JCR) large artery compliance in JCR rats. 20-HETE antagonists also decreased systolic (182 ± 3 mmHg JCR, 145 ± 3 mmHg JCR + 20-HETE antagonists) but not diastolic blood pressure in JCR rats. Whereas diastolic pressure was fully angiotensin II (Ang II)-dependent, systolic pressure was only partially Ang II-dependent, and large artery stiffness was Ang II-independent. Thus, 20-HETE-dependent regulation of systolic blood pressure may be a unique feature of metabolic syndrome related to high 20-HETE production in large, conduit arteries, which results in increased large artery stiffness and systolic blood pressure. These findings may have implications for management of systolic hypertension in patients with metabolic syndrome.

Cyp2c44 gene disruption is associated with increased hematopoietic stem cells: implication in chronic hypoxia-induced pulmonary hypertension

We have recently demonstrated that disruption of the murine cytochrome P-450 2c44 gene (Cyp2c44) exacerbates chronic hypoxia-induced pulmonary artery remodeling and hypertension in mice. Subsequently, we serendipitously found that Cyp2c44 gene disruption also increases hematopoietic stem cell (HSC) numbers in bone marrow and blood. Therefore, the objective of the present study was to investigate whether CYP2C44-derived eicosanoids regulate HSC proliferation/cell growth and whether increased HSCs contribute to chronic hypoxia-induced remodeling of pulmonary arteries in Cyp2c44 knockout mice. Our findings demonstrated that lack of CYP2C44 epoxygenase, which catalyzed the oxidation of arachidonic acid to epoxyeicosatrienoic (EETs) and hydroxyeicosatetraenoic (HETE) acids, increases the numbers of 1) HSCs (CD34+, CD117+, and CD133+), 2) proangiogenic (CD34+CD133+ and CD34+CD117+CD133+) cells, and 3) immunogenic/inflammatory (CD34+CD11b+, CD133+CD11b+, F4/80+, CD11b+, and F4/80+CD11b+) macrophages in bone marrow and blood compared with wild-type mice. Among the various CYP2C44-derived arachidonic acids, only 15-HETE decreased CD117+ cell numbers when applied to bone marrow cell cultures. Interestingly, CD133+ and von Willebrand factor-positive cells, which are derived from proangiogenic stem cells, are increased in the bone marrow, blood, and lungs of mice exposed to chronic hypoxia and in remodeled and occluded pulmonary arteries of CYP2C44-deficient mice. In conclusion, our results demonstrate that CYP2C44-derived 15-HETE plays a critical role in downregulating HSC proliferation and growth, because disruption of the Cyp2c44 gene increased HSCs that potentially contribute to chronic hypoxia-induced pulmonary arterial remodeling and occlusion.NEW & NOTEWORTHY This study demonstrates that cytochrome P-450 2C44 plays a critical role in controlling the phenotype of hematopoietic stem cells and that when this enzyme is knocked out, stem cells are differentiated. These stem cells give rise to increased circulating monocytes and macrophages and contribute to the pathogenesis of chronic hypoxia-induced pulmonary artery remodeling and hypertension.