Jalees Rehman

Secretion of Angiogenic and Antiapoptotic Factors by Human Adipose Stromal Cells

Background—The delivery of autologous cells to increase angiogenesis is emerging as a treatment option for patients with cardiovascular disease but may be limited by the accessibility of sufficient cell numbers. The beneficial effects of delivered cells appear to be related to their pluripotency and ability to secrete growth factors. We examined nonadipocyte stromal cells from human subcutaneous fat as a novel source of therapeutic cells. Methods and Results—Adipose stromal cells (ASCs) were isolated from human subcutaneous adipose tissue and characterized by flow cytometry. ASCs secreted 1203±254 pg of vascular endothelial growth factor (VEGF) per 106 cells, 12 280±2944 pg of hepatocyte growth factor per 106 cells, and 1247±346 pg of transforming growth factor-&bgr; per 106 cells. When ASCs were cultured in hypoxic conditions, VEGF secretion increased 5-fold to 5980±1066 pg/106 cells (P =0.0016). The secretion of VEGF could also be augmented 200-fold by transfection of ASCs with a plasmid encoding VEGF (P <0.05). Conditioned media obtained from hypoxic ASCs significantly increased endothelial cell growth (P <0.001) and reduced endothelial cell apoptosis (P <0.05). Nude mice with ischemic hindlimbs demonstrated marked perfusion improvement when treated with human ASCs (P <0.05). Conclusions—Our experiments delineate the angiogenic and antiapoptotic potential of easily accessible subcutaneous adipose stromal cells by demonstrating the secretion of multiple potentially synergistic proangiogenic growth factors. These findings suggest that autologous delivery of either native or transduced subcutaneous ASCs, which are regulated by hypoxia, may be a novel therapeutic option to enhance angiogenesis or achieve cardiovascular protection.

Peripheral blood "endothelial progenitor cells" are derived from monocyte/macrophages and secrete angiogenic growth factors

Background—Endothelial progenitor cells (EPCs) have been isolated from peripheral blood and can enhance angiogenesis after infusion into host animals. It is not known whether the proangiogenic effects are a result of such events as endothelial differentiation and subsequent proliferation of EPCs or secondary to secretion of angiogenic growth factors. Methods and Results—Human EPCs were isolated as previously described, and their phenotypes were confirmed by uptake of acetylated LDL and binding of ulex-lectin. EPC proliferation and surface marker expression were analyzed by flow cytometry, and conditioned medium was assayed for growth factors. The majority of EPCs expressed monocyte/macrophage markers such as CD14 (95.7±0.3%), Mac-1 (57.6±13.5%), and CD11c (90.8±4.9%). A much lower percentage of cells expressed the specific endothelial marker VE-cadherin (5.2±0.7%) or stem/progenitor-cell markers AC133 (0.16±0.05%) and c-kit (1.3±0.7%). Compared with circulating monocytes, cultured EPCs showed upregulation of monocyte activation and macrophage differentiation markers. EPCs did not demonstrate any significant proliferation but did secrete the angiogenic growth factors vascular endothelial growth factor, hepatocyte growth factor, granulocyte colony–stimulating factor, and granulocyte-macrophage colony–stimulating factor. Conclusions—Our findings suggest that acetylated LDL(+)ulex-lectin(+) cells, commonly referred to as EPCs, do not proliferate but release potent proangiogenic growth factors. The majority of acetylated LDL(+)ulex-lectin(+) cells are derived from monocyte/macrophages. The findings of low proliferation and endothelial differentiation suggest that their angiogenic effects are most likely mediated by growth factor secretion. These findings may allow for development of novel angiogenic therapies relying on secreted growth factors or on recruitment of endogenous monocytes/macrophages to sites of ischemia.

Epigenetic Attenuation of Mitochondrial Superoxide Dismutase 2 in Pulmonary Arterial Hypertension A Basis for Excessive Cell Proliferation and a New Therapeutic Target

Background— Excessive proliferation and impaired apoptosis of pulmonary artery (PA) smooth muscle cells (PASMCs) contribute to vascular obstruction in patients and fawn-hooded rats (FHRs) with PA hypertension (PAH). Expression and activity of mitochondrial superoxide dismutase-2 (SOD2), the major generator of H2O2, is known to be reduced in PAH; however, the mechanism and therapeutic relevance of this are unknown. Methods and Results— SOD2 expression in PASMCs is decreased in PAH patients and FHRs with PAH. FHR PASMCs have higher proliferation and lower apoptosis rates than Sprague-Dawley rat PASMCs. Moreover, FHR PASMCs have hyperpolarized mitochondria, low H2O2 production, and reduced cytoplasmic and mitochondrial redox state. Administration of SOD2 small interfering RNA to normal PASMCs recapitulates the FHR PAH phenotype, hyperpolarizing mitochondria, decreasing H2O2, and inhibiting caspase activity. Conversely, SOD2 overexpression in FHR PASMCs or therapy with the SOD-mimetic metalloporphyrin Mn(III)tetrakis (4-benzoic acid) porphyrin (MnTBAP) reverses the hyperproliferative PAH phenotype. Importantly, SOD-mimetic therapy regresses PAH in vivo. Investigation of the SOD2 gene revealed no mutation, suggesting a possible epigenetic dysregulation. Genomic bisulfite sequencing demonstrates selective hypermethylation of a CpG island in an enhancer region of intron 2 and another in the promoter. Differential methylation occurs selectively in PAs versus aortic SMCs and is reversed by the DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine, restoring both SOD2 expression and the ratio of proliferation to apoptosis. Expression of the enzymes that mediate gene methylation, DNA methyltransferases 1 and 3B, is upregulated in FHR lungs. Conclusions— Tissue-specific, epigenetic SOD2 deficiency initiates and sustains a heritable form of PAH by impairing redox signaling and creating a proliferative, apoptosis-resistant PASMC. SOD augmentation regresses experimental PAH. The discovery of an epigenetic component to PAH may offer new therapeutic targets.

Inhibition of mitochondrial fission prevents cell cycle progression in lung cancer

Mitochondria exist in dynamic networks that undergo fusion and fission. Mitochondrial fusion and fission are mediated by several GTPases in the outer mitochondrial membrane, notably mitofusin‐2 (Mfn‐2), which promotes fusion, and dynamin‐related protein (Drp‐1), which promotes fission. We report that human lung cancer cell lines exhibit an imbalance of Drp‐1/Mfn‐2 expression, which promotes a state of mitochondrial fission. Lung tumor tissue samples from patients demonstrated a similar increase in Drp‐1 and decrease in Mfn‐2 when compared to adjacent healthy lung. Complementary approaches to restore mitochondrial network formation in lung cancer cells by overexpression of Mfn‐2, Drp‐1 inhibition, or Drp‐1 knockdown resulted in a marked reduction of cancer cell proliferation and an increase in spontaneous apoptosis. The number of cancer cells in S phase decreased from 32.4 ± 0.6 to 6.4 ± 0.3% with Drp‐1 inhibition (P< 0.001). In a xenotransplantation model, Mfn‐2 gene therapy or Drp‐1 inhibition could regress tumor growth. The tumor volume decreased from 205.6 ± 59 to 70.6 ± 15 mm3 (P<0.05) with Mfn‐2 overexpression and from 186.0 ± 19 to 87.0 ± 6 mm3 (P<0.01) with therapeutic Drp‐1 inhibition. Impaired fusion and enhanced fission contribute fundamentally to the proliferation/apoptosis imbalance in cancer and constitute promising novel therapeutic targets.—Rehman, J., Zhang, H. J., Toth, P. T., Zhang, Y., Marsboom, G., Hong, Z., Salgia, R., Husain, A. N., Wietholt, C., Archer, S. L. Inhibition of mitochondrial fission prevents cell cycle progression in lung cancer. FASEB J. 26, 2175‐2186 (2012). www.fasebj.org

Dynamin-Related Protein 1–Mediated Mitochondrial Mitotic Fission Permits Hyperproliferation of Vascular Smooth Muscle Cells and Offers a Novel Therapeutic Target in Pulmonary Hypertension

Rationale: Pulmonary arterial hypertension (PAH) is a lethal syndrome characterized by pulmonary vascular obstruction caused, in part, by pulmonary artery smooth muscle cell (PASMC) hyperproliferation. Mitochondrial fragmentation and normoxic activation of hypoxia-inducible factor-1&agr; (HIF-1&agr;) have been observed in PAH PASMCs; however, their relationship and relevance to the development of PAH are unknown. Dynamin-related protein-1 (DRP1) is a GTPase that, when activated by kinases that phosphorylate serine 616, causes mitochondrial fission. It is, however, unknown whether mitochondrial fission is a prerequisite for proliferation. Objective: We hypothesize that DRP1 activation is responsible for increased mitochondrial fission in PAH PASMCs and that DRP1 inhibition may slow proliferation and have therapeutic potential. Methods and Results: Experiments were conducted using human control and PAH lungs (n=5) and PASMCs in culture. Parallel experiments were performed in rat lung sections and PASMCs and in rodent PAH models induced by the HIF-1&agr; activator, cobalt, chronic hypoxia, and monocrotaline. HIF-1&agr; activation in human PAH leads to mitochondrial fission by cyclin B1/CDK1–dependent phosphorylation of DRP1 at serine 616. In normal PASMCs, HIF-1&agr; activation by CoCl2 or desferrioxamine causes DRP1-mediated fission. HIF-1&agr; inhibition reduces DRP1 activation, prevents fission, and reduces PASMC proliferation. Both the DRP1 inhibitor Mdivi-1 and siDRP1 prevent mitotic fission and arrest PAH PASMCs at the G2/M interphase. Mdivi-1 is antiproliferative in human PAH PASMCs and in rodent models. Mdivi-1 improves exercise capacity, right ventricular function, and hemodynamics in experimental PAH. Conclusions: DRP-1–mediated mitotic fission is a cell-cycle checkpoint that can be therapeutically targeted in hyperproliferative disorders such as PAH.

The inhibition of pyruvate dehydrogenase kinase improves impaired cardiac function and electrical remodeling in two models of right ventricular hypertrophy: resuscitating the hibernating right ventricle

Right ventricular hypertrophy (RVH) and RV failure contribute to morbidity and mortality in pulmonary arterial hypertension (PAH). The cause of RV dysfunction and the feasibility of therapeutically targeting the RV are uncertain. We hypothesized that RV dysfunction and electrical remodeling in RVH result, in part, from a glycolytic shift in the myocyte, caused by activation of pyruvate dehydrogenase kinase (PDK). We studied two complementary rat models: RVH + PAH (induced by monocrotaline) and RVH + without PAH (induced by pulmonary artery banding (PAB)). Monocrotaline RVH reduced RV O2-consumption and enhanced glycolysis. RV 2-fluoro-2-deoxy-glucose uptake, Glut-1 expression, and pyruvate dehydrogenase phosphorylation increased in monocrotaline RVH. The RV monophasic action potential duration and QTc interval were prolonged due to decreased expression of repolarizing voltage-gated K+ channels (Kv1.5, Kv4.2). In the RV working heart model, the PDK inhibitor, dichloroacetate, acutely increased glucose oxidation and cardiac work in monocrotaline RVH. Chronic dichloroacetate therapy improved RV repolarization and RV function in vivo and in the RV Langendorff model. In PAB-induced RVH, a similar reduction in cardiac output and glycolytic shift occurred and it too improved with dichloroacetate. In PAB-RVH, the benefit of dichloroacetate on cardiac output was approximately 1/3 that in monocrotaline RVH. The larger effects in monocrotaline RVH likely reflect dichloroacetate’s dual metabolic benefits in that model: regression of vascular disease and direct effects on the RV. Reduction in RV function and electrical remodeling in two models of RVH relevant to human disease (PAH and pulmonic stenosis) result, in part, from a PDK-mediated glycolytic shift in the RV. PDK inhibition partially restores RV function and regresses RVH by restoring RV repolarization and enhancing glucose oxidation. Recognition that a PDK-mediated metabolic shift contributes to contractile and ionic dysfunction in RVH offers insight into the pathophysiology and treatment of RVH.

Empowering self-renewal and differentiation: the role of mitochondria in stem cells

Stem cells are characterized by their multi-lineage differentiation potential (pluripotency) and their ability for self-renewal, which permits them to proliferate while avoiding lineage commitment and senescence. Recent studies demonstrate that undifferentiated, pluripotent stem cells display lower levels of mitochondrial mass and oxidative phosphorylation, and instead preferentially use non-oxidative glycolysis as a major source of energy. Hypoxia is a potent suppressor of mitochondrial oxidation and appears to promote “stemness” in adult and embryonic stem cells. This has lead to an emerging paradigm, that mitochondrial oxidative metabolism is not just an indicator of the undifferentiated state of stem cells, but may also regulate the pluripotency and self-renewal of stem cells. The identification of specific mitochondrial pathways that regulate stem cell fate may therefore enable metabolic programming and reprogramming of stem cells.

Obesity is associated with increased levels of circulating hepatocyte growth factor

OBJECTIVES This study evaluated whether obesity in humans was associated with an increase in circulating hepatocyte growth factor (HGF) and vascular endothelial growth factor (VEGF) levels. BACKGROUND Obesity acts as a cardiovascular risk factor by mechanisms that are not fully understood. Adipose tissue is able to secrete multiple cytokines and growth factors ex vivo. We hypothesized that the increased presence of adipose tissue in obese subjects results in systemic elevations of the mitogenic factors HGF and VEGF. METHODS Blood samples were obtained from lean (n = 21) and obese (n = 44) volunteers. Serum HGF and VEGF levels were assessed by enzyme-linked immunoadsorbent assay. Insulin and fasting glucose levels were measured to evaluate insulin sensitivity. Conditioned medium of adipose cells was assayed for HGF secretion. RESULTS Serum HGF levels in obese subjects were more than three-fold higher than those of lean subjects (2,462 +/- 184 pg/ml vs. 765 +/- 48 pg/ml, p < 0.0001). The VEGF levels were not significantly elevated in obese subjects (135 +/- 31 pg/ml vs. 128 +/- 37 pg/ml). The HGF concentrations, but not VEGF concentrations, were significantly correlated with body mass index (BMI) (p < 0.0001, r = 0.74). The observed increases in HGF concentrations of obese subjects were not secondary to insulin resistance or hypertension. Freshly isolated human adipose cells secreted HGF. CONCLUSIONS Our results indicate that obesity is associated with a marked increase in circulating HGF levels, which correlate linearly with BMI. Because vascular growth factors have been associated with the pathogenesis of atherosclerosis, the possible role of such humoral factors as a link between obesity and cardiovascular disease is very intriguing.

Increased production of antigen-specific immunoglobulins G and M following in vivo treatment with the medicinal plants Echinacea angustifolia and Hydrastis canadensis.

A number of immunomodulatory effects have been attributed to the medicinal plants Echinacea angustifolia and Goldenseal (Hydrastis canadensis); however, little is known about whether treatment with these plants can enhance antigen-specific immunity. We investigated the antigen-specific in vivo immunomodulatory potential of continuous treatment with Echinacea and Goldenseal root extract over a period of 6 weeks using rats that were injected with the novel antigen keyhole limpet hemocyanin (KLH) and re-exposed to KLH after the initial exposure. Immunoglobulin production was monitored via ELISA continuously over a period of 6 weeks. The Echinacea-treated group showed a significant augmentation of their primary and secondary IgG response to the antigen, whereas the Goldenseal-treated group showed an increase in the primary IgM response during the first 2 weeks of treatment. Our results suggest that medicinal plants like Echinacea or Goldenseal may enhance immune function by increasing antigen-specific immunoglobulin production.

Premature senescence of highly proliferative endothelial progenitor cells is induced by tumor necrosis factor-α via the p38 mitogen-activated protein kinase pathway

Senescence of endothelial cells increases with systemic aging and is thought to contribute to the development of atherosclerosis. Cell therapy with highly proliferative endothelial progenitor cells (EPCs) is an emerging therapeutic option to promote endothelial regeneration, but little is known about their senescence and their vulnerability to inflammatory stressors. We therefore studied the senescence of proliferative human EPCs and investigated the effects of the proinflammatory cytokine tumor necrosis factor‐α (TNF‐α) on their senescence. Human EPCs had a significantly lower rate of senescence at baseline, compared with that of mature endothelial cells. However, EPCs up‐regulated the expression of the senescence‐associated cell cycle arrest protein p16INK4aand markedly increased measured senescence levels when exposed to chronic TNF‐α treatment. Analysis of telomere length showed that the increases in senescence were not related to changes in telomere length. Inhibition of the p38 mitogen‐activated protein kinase pathway blocked the induction of p16INK4a and cellular senescence. In conclusion, highly proliferative EPCs have a low rate of intrinsic senescence but are vulnerable to premature senescence induction by chronic proinflammatory stimulation. These findings will lead to a better understanding of physiological endothelial regeneration as well as to targeted therapies with the aim of promoting endothelial regeneration through endothelial progenitor cells.— Zhang, Y., Herbert, B.‐S., Rajashekhar, G., Ingram, D. A., Yoder, M. C., Clauss, M., Rehman. J. Premature senescence of highly proliferative endothelial progenitor cells is induced by tumor necrosis factor‐α via the p38 mitogen‐activated protein kinase pathway. FASEBJ. 23, 1358–1365 (2009)