Pluripotent hematopoietic stem cells augment α-adrenergic receptor-mediated contraction of pulmonary artery and contribute to the pathogenesis of pulmonary hypertension.

Pulmonary hypertension (PH) is a multicellular and progressive disease with a high mortality rate. Among many cell types, hematopoietic stem cells (HSCs) are incriminated in the pathogenesis of PH. However, our understanding of the mechanisms that increase HSCs in blood and lungs of hypertensive animals or patients and the role played by HSCs in the pathogenesis of PH remains elusive. Studies suggest that glycolysis is critical for the survival and growth of HSCs. In various cell types from hypertensive lungs of animals and patients, glycolysis and the glucose-6-phosphate dehydrogenase (G6PD) activity are increased. Herein, we demonstrated in mice that chronic hypoxia increased HSCs (CD34+, CD117+, CD133+, CD34+/CD117+, and CD34+/CD133+) in bone marrow and blood and around hypertensive pulmonary arteries in a time-dependent manner. Intriguingly, we found fewer CD133+ cells in the bone marrow of C57BL/6 mice compared with Sv129J mice, and C57BL mice developed less severe chronic hypoxia-elicited PH and heart failure than Sv129J mice. Similarly, the numbers of CD34+ and CD117+ cells in blood of patients with pulmonary arterial hypertension (PAH) were higher (>3-fold) compared with healthy individuals. By allogeneic bone marrow transplantation, we found that GFP+ bone marrow cells infiltrated the lungs and accumulated around the pulmonary arteries in lungs of hypoxic mice, and these cells contributed to increased α-adrenergic receptor-mediated contraction of the pulmonary artery cultured in hypoxia. Inhibition of G6PD activity with (3ß,5α)-3,21-dihydroxypregnan-20-one, a novel and potent G6PD inhibitor, decreased HSCs in bone marrow, blood, and lungs of hypoxic mice and reduced α-agonist-induced contraction of the pulmonary artery and established hypoxia-induced PH. We did not observe CD133+ cells around the pulmonary arteries in the lungs of chronically hypoxic G6PD-deficient mice. Furthermore, knockdown of G6PD and inhibition of G6PD activity: 1) downregulated canonical and noncanonical Wnt and Fzd receptors genes; 2) upregulated Bmpr1a; 3) decreased Cxcl12, and 4) reduced HSC (CD117+ and CD133+) numbers. In all, our findings demonstrate unexpected function for bone marrow-derived HSCs in augmenting α-adrenergic receptor-mediated contraction of pulmonary arteries and remodeling of pulmonary arteries that contribute to increase pulmonary vascular resistance in PAH patients and hypoxic mice and suggest that G6PD, by regulating expression of genes in the WNT and BMPR signaling, contributed to increase and release of HSCs from the bone marrow in response to hypoxic stimuli.

Accelerated cardiomyocyte senescence contributes to late-onset doxorubicin-induced cardiotoxicity.

Children surviving cancer and chemotherapy are at risk for adverse health events including heart failure that may be delayed by years. Although the early effects of doxorubicin-induced cardiotoxicity may be attributed to a direct effect on the cardiomyocytes, the mechanisms underlying the delayed or late effects (8-20 yr) are unknown. The goal of this project was to develop a model of late-onset doxorubicin-induced cardiotoxicity to better delineate the underlying pathophysiology responsible. The underlying hypothesis was that doxorubicin-induced "late-onset cardiotoxicity" was the result of mitochondrial dysfunction leading to cell failure and death. Wistar rats, 3-4 wk of age, were randomly assigned to vehicle or doxorubicin injection groups (1-45 mg/kg). Cardiovascular function was unaltered at the lower dosages (1-15 kg/mg), but beginning at 6 mo after injection significant cardiac degradation was observed in the 45 mg/kg group. Doxorubicin significantly increased myocardial mitochondrial DNA (mtDNA) damage. In contrast, in isolated c-kit left ventricular (LV) cells, doxorubicin treatment did not increase mtDNA damage. Biomarkers of senescence within the LV were significantly increased, suggesting accelerated aging of the LV. Doxorubicin also significantly increased LV histamine content suggestive of mast cell activation. With the use of flow cytometry, a significant expansion of the c-kit and stage-specific embryonic antigen 1 cell populations within the LV were concomitant with significant decreases in the circulating peripheral blood population of these cells. These results are consistent with the concept that doxorubicin induced significant damage to the cardiomyocyte population and that although the heart attempted to compensate it eventually succumbed to an inability for self-repair.

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.

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.

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 18ß-glycyrrhetinic acid (18ß-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 18ß-GA and Gap-27. Application of 15-HETE and 20-HETE to BIPAs increased SM-MHC expression, which was also suppressed by 18ß-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 18ß-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.

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.

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.

Nucleosome dynamics: HMGB1 relaxes canonical nucleosome structure to facilitate estrogen receptor binding.

High mobility group protein 1 (HMGB1) interacts with DNA and chromatin to influence the regulation of transcription, DNA repair and recombination. We show that HMGB1 alters the structure and stability of the canonical nucleosome (N) in a nonenzymatic, ATP-independent manner. Although estrogen receptor (ER) does not bind to its consensus estrogen response element within a nucleosome, HMGB1 restructures the nucleosome to facilitate strong ER binding. The isolated HMGB1-restructured nucleosomes (N' and N″) remain stable and exhibit characteristics distinctly different from the canonical nucleosome. These findings complement previous studies that showed (i) HMGB1 stimulates in vivo transcriptional activation at estrogen response elements and (ii) knock down of HMGB1 expression by siRNA precipitously reduced transcriptional activation. The findings indicate that one aspect of the mechanism of HMGB1 action involves a restructuring of the nucleosome that appears to relax structural constraints within the nucleosome.