Junya Azuma

Safety Evaluation of Clinical Gene Therapy Using Hepatocyte Growth Factor to Treat Peripheral Arterial Disease

Therapeutic angiogenesis using angiogenic growth factors is expected to be a new treatment for patients with critical limb ischemia (CLI). Because hepatocyte growth factor (HGF) has potent angiogenic activity, we investigated the safety and efficiency of HGF plasmid DNA in patients with CLI as a prospective open-labeled clinical trial. Intramuscular injection of naked HGF plasmid DNA was performed in ischemic limbs of 6 CLI patients with arteriosclerosis obliterans (n= 3) or Buerger disease (n= 3) graded as Fontaine III or IV. The primary end points were safety and improvement of ischemic symptoms at 12 weeks after transfection. Severe complications and adverse effects caused by gene transfer were not detected in any patients. Of particular importance, no apparent edema was observed in any patient throughout the trial. In addition, serum HGF concentration was not changed throughout the therapy period in all patients. In contrast, a reduction of pain scale of more than 1 cm in visual analog pain scale was observed in 5 of 6 patients. Increase in ankle pressure index more than 0.1 was observed in 5 of 5 patients. The long diameter of 8 of 11 ischemic ulcers in 4 patients was reduced > 25%. Intramuscular injection of naked HGF plasmid is safe, feasible, and can achieve successful improvement of ischemic limbs. Although the present data are conducted to demonstrate the safety as phase I/early phase IIa, the initial clinical outcome with HGF gene transfer seems to indicate usefulness as sole therapy for CLI.

Inhibition of microRNA-29b reduces murine abdominal aortic aneurysm development

MicroRNAs (miRs) regulate gene expression at the posttranscriptional level and play crucial roles in vascular integrity. As such, they may have a role in modifying abdominal aortic aneurysm (AAA) expansion, the pathophysiological mechanisms of which remain incompletely explored. Here, we investigate the role of miRs in 2 murine models of experimental AAA: the porcine pancreatic elastase (PPE) infusion model in C57BL/6 mice and the AngII infusion model in Apoe-/- mice. AAA development was accompanied by decreased aortic expression of miR-29b, along with increased expression of known miR-29b targets, Col1a1, Col3a1, Col5a1, and Eln, in both models. In vivo administration of locked nucleic acid anti-miR-29b greatly increased collagen expression, leading to an early fibrotic response in the abdominal aortic wall and resulting in a significant reduction in AAA progression over time in both models. In contrast, overexpression of miR-29b using a lentiviral vector led to augmented AAA expansion and significant increase of aortic rupture rate. Cell culture studies identified aortic fibroblasts as the likely vascular cell type mediating the profibrotic effects of miR-29b modulation. A similar pattern of reduced miR-29b expression and increased target gene expression was observed in human AAA tissue samples compared with that in organ donor controls. These data suggest that therapeutic manipulation of miR-29b and its target genes holds promise for limiting AAA disease progression and protecting from rupture.

Apelin is necessary for the maintenance of insulin sensitivity

The recently discovered peptide apelin is known to be involved in the maintenance of insulin sensitivity. However, questions persist regarding its precise role in the chronic setting. Fasting glucose, insulin, and adiponectin levels were determined on mice with generalized deficiency of apelin (APKO). Additionally, insulin (ITT) and glucose tolerance tests (GTT) were performed. To assess the impact of exogenously delivered apelin on insulin sensitivity, osmotic pumps containing pyroglutamated apelin-13 or saline were implanted in APKO mice for 4 wk. Following the infusion, ITT/GTTs were repeated and the animals euthanized. Soleus muscles were harvested and homogenized in lysis buffer, and insulin-induced Akt phosphorylation was determined by Western blotting. Apelin-13 infusion and ITTs/GTTs were also performed in obese diabetic db/db mice. To probe the underlying mechanism for apelins effects, apelin-13 was also delivered to cultured C2C12 myotubes. 2-[3H]deoxyglucose uptake and Akt phosphorylation were assessed in the presence of various inhibitors. APKO mice had diminished insulin sensitivity, were hyperinsulinemic, and had decreased adiponectin levels. Soleus lysates had decreased insulin-induced Akt phosphorylation. Administration of apelin to APKO and db/db mice resulted in improved insulin sensitivity. In C2C12 myotubes, apelin increased glucose uptake and Akt phosphorylation. These events were fully abrogated by pertussis toxin, compound C, and siRNA knockdown of AMPKalpha1 but only partially diminished by LY-294002 and not at all by L-NAME. We conclude that apelin is necessary for the maintenance of insulin sensitivity in vivo. Apelins effects on glucose uptake and Akt phosphorylation are in part mediated by a G(i) and AMPK-dependent pathway.

MicroRNA-21 Blocks Abdominal Aortic Aneurysm Development and Nicotine-Augmented Expansion

miR-21 modulates abdominal aortic aneurysm development by regulating cell proliferation and apoptosis within the aortic wall. miR-21, a Red Alert for AAA Abdominal aortic aneurysms (AAAs) constitute a major public health burden, with few treatment options. In this common condition associated with increased age, male gender, high blood pressure, and especially smoking, the major conduit vessel within the abdomen slowly enlarges and may rupture, often fatally. MicroRNAs are short molecules that can simultaneously regulate translation of multiple genes. One example, microRNA-21 (miR-21), has been shown to control gene expression patterns that influence a variety of cellular processes including maturation, migration, proliferation, and survival. In a new study, Maegdefessel et al. investigated the role of miR-21 in two well-established mouse models of AAA: one in which the aorta is exposed to enzymatic degradation of supporting tissue and another in which mice predisposed to vascular disease spontaneously form AAA in response to the peptide hormone angiotensin II. In both models, miR-21 expression increased within the aortic wall as the AAA developed. miR-21 was also elevated in samples of aorta from patients with AAA compared with healthy controls. Nicotine, the major constituent of tobacco, accelerated AAA growth in both mouse models and caused an even larger increase in miR-21 expression. This appeared to be a protective response because preventing an increase in miR-21 with an inhibitor increased AAA growth and rupture rates in both models. In contrast, exogenous supplementation of miR-21 slowed aneurysm growth and prevented rupture, even in the presence of nicotine. This was partly mediated through miR-21’s suppressive effects on the protein PTEN (phosphatase and tensin homolog). Cell culture studies demonstrated that inflammatory stimuli, known to influence AAA development, increased miR-21 expression. These results suggest that enhanced miR-21 expression is an endogenous response to pathological aortic dilation and may offer a new therapeutic pathway that could be targeted to treat AAA in patients. Identification and treatment of abdominal aortic aneurysm (AAA) remains among the most prominent challenges in vascular medicine. MicroRNAs are crucial regulators of cardiovascular pathology and represent possible targets for the inhibition of AAA expansion. We identified microRNA-21 (miR-21) as a key modulator of proliferation and apoptosis of vascular wall smooth muscle cells during development of AAA in two established murine models. In both models (AAA induced by porcine pancreatic elastase or infusion of angiotensin II), miR-21 expression increased as AAA developed. Lentiviral overexpression of miR-21 induced cell proliferation and decreased apoptosis in the aortic wall, with protective effects on aneurysm expansion. miR-21 overexpression substantially decreased expression of the phosphatase and tensin homolog (PTEN) protein, leading to increased phosphorylation and activation of AKT, a component of a pro-proliferative and antiapoptotic pathway. Systemic injection of a locked nucleic acid–modified antagomir targeting miR-21 diminished the pro-proliferative impact of down-regulated PTEN, leading to a marked increase in the size of AAA. Similar results were seen in mice with AAA augmented by nicotine and in human aortic tissue samples from patients undergoing surgical repair of AAA (with more pronounced effects observed in smokers). Modulation of miR-21 expression shows potential as a new therapeutic option to limit AAA expansion and vascular disease progression.

Therapeutic angiogenesis using hepatocyte growth factor (HGF).

HGF is a mesenchyme-derived pleiotropic factor, which regulates cell growth, cell motility, and morphogenesis of various types of cells and is thus considered a humoral mediator of epithelial-mesenchymal interactions responsible for morphogenic tissue interactions during embryonic development and organogenesis. Although HGF was originally identified as a potent mitogen for hepatocytes, it has also been identified as a member of angiogenic growth factors. Interestingly, the presence of its specific receptor, c-met, is observed in vascular cells and cardiac myocytes. In addition, among growth factors, the mitogenic action of HGF on human endothelial cells was most potent. Recent studies have demonstrated the potential application of HGF to treat cardiovascular diseases such as peripheral vascular disease, myocardial infarction and cerebrovascular disease. In this review, we will discuss a potential therapeutic strategy using HGF in cardiovascular disease.

Phase I/IIa clinical trial of therapeutic angiogenesis using hepatocyte growth factor gene transfer to treat critical limb ischemia.

Objective—To evaluate the safety and feasibility of intramuscular gene transfer using naked plasmid DNA-encoding hepatocyte growth factor (HGF) and to assess its potential therapeutic benefit in patients with critical limb ischemia. Methods and Results—Gene transfer was performed in 22 patients with critical limb ischemia by intramuscular injection of HGF plasmid, either 2 or 4 mg, 2 times. Safety, ankle-brachial index, resting pain on a 10-cm visual analog scale, wound healing, and walking distance were evaluated before treatment and at 2 months after injection. No serious adverse event caused by gene transfer was detected over a follow-up of 6 months. Of particular importance, no peripheral edema, in contrast to that seen after treatment with vascular endothelial growth factor, was observed. In addition, the systemic HGF protein level did not increase during the study. At 2 months after gene transfer, the mean±SD ankle-brachial index increased from 0.46±0.08 to 0.59±0.13 (P<0.001), the mean±SD size of the largest ischemic ulcers decreased from 3.08±1.54 to 2.32±1.88 cm (P=0.007), and the mean±SD visual analog scale score decreased from 5.92±1.67 to 3.04±2.50 cm (P=0.001). An increase in ankle-brachial index by >0.1, a reduction in ulcer size by >25%, and a reduction in visual analog scale score by >2 cm at 2 months after gene transfer were observed in 11 (64.7%) of 17 limbs, 18 (72%) of 25 ulcers, and 8 (61.5%) of 13 limbs, respectively. Conclusion—Intramuscular injection of naked HGF plasmid is safe and feasible and can achieve successful improvement of ischemic limbs as sole therapy.

miR-24 limits aortic vascular inflammation and murine abdominal aneurysm development

Identification and treatment of abdominal aortic aneurysm (AAA) remain among the most prominent challenges in vascular medicine. MicroRNAs (miRNAs) are crucial regulators of cardiovascular pathology and represent intriguing targets to limit AAA expansion. Here we show, by using two established murine models of AAA disease along with human aortic tissue and plasma analysis, that miR-24 is a key regulator of vascular inflammation and AAA pathology. In vivo and in vitro studies reveal chitinase 3-like 1 (Chi3l1) to be a major target and effector under the control of miR-24, regulating cytokine synthesis in macrophages as well as their survival, promoting aortic smooth muscle cell migration and cytokine production, and stimulating adhesion molecule expression in vascular endothelial cells. We further show that modulation of miR-24 alters AAA progression in animal models, and that miR-24 and CHI3L1 represent novel plasma biomarkers of AAA disease progression in humans.

Novel Mechanisms of Valsartan on the Treatment of Acute Myocardial Infarction Through Inhibition of the Antiadhesion Molecule Periostin

Our previous study demonstrated that periostin, an extracellular matrix protein, plays an important role in left ventricular remodeling through the inhibition of cell–cell interactions. Because the gene regulation of periostin has not yet been examined, we focused on the effects of angiotensin (Ang) II and mechanical stretch, because Ang II and mechanical stretch are related to cardiac remodeling after myocardial infarction. First, we examined the effects of Ang II on periostin in myocytes and fibroblasts in vitro. Ang II significantly increased periostin through phosphatidylinositol 3-kinase, c-Jun N-terminal kinase, p38, and extracellular signal-regulated kinase 1/2 pathways in myocytes and fibroblasts (P<0.05). On the other hand, mechanical stretch also significantly increased periostin expression (P<0.05). This increase was inhibited partially, but significantly, by an Ang II receptor blocker, valsartan, and inhibited almost completely by valsartan with the neutralization antibodies for transforming growth factor-&bgr; and platelet-derived growth factor–BB (P<0.05). Therefore, we further examined periostin expression in vivo. Periostin expression was significantly increased in infarcted myocardium (P<0.05), and treatment with valsartan significantly attenuated it at 4 weeks after myocardial infarction (P<0.05), accompanied by a significant improvement in cardiac dysfunction (P<0.05). Overall, the present study demonstrated that Ang II, as well as mechanical stretch, stimulated periostin expression in both cardiac myocytes and fibroblasts, whereas valsartan significantly attenuated the increase in periostin expression. The inhibition of periostin by valsartan might especially contribute to its beneficial effects on cardiac remodeling after myocardial infarction.

Hepatocyte Growth Factor, but not Vascular Endothelial Growth Factor, Attenuates Angiotensin II–Induced Endothelial Progenitor Cell Senescence

Although both hepatocyte growth factor (HGF) and vascular endothelial growth factor (VEGF) are potent angiogenic growth factors in animal models of ischemia, their characteristics are not the same in animal experiments and clinical trials. To elucidate the discrepancy between HGF and VEGF, we compared the effects of HGF and VEGF on endothelial progenitor cells under angiotensin II stimulation, which is a well-known risk factor for atherosclerosis. Here, we demonstrated that HGF, but not VEGF, attenuated angiotensin II–induced senescence of endothelial progenitor cells through a reduction of oxidative stress by inhibition of the phosphatidylinositol-3,4,5-triphosphate/rac1 pathway. Potent induction of neovascularization of endothelial progenitor cells by HGF, but not VEGF, under angiotensin II was also confirmed by in vivo experiments using several models, including HGF transgenic mice.

Hepatocyte Growth Factor Reduces Cardiac Fibrosis by Inhibiting Endothelial-Mesenchymal Transition

The purpose of this study was to investigate the effect of hepatocyte growth factor (HGF) on the pathogenesis of cardiac fibrosis induced by pressure overload in mice. Although cardiac fibrosis is attributed to excess pathological deposition of extracellular matrix components, the mechanism remains unclear. Recent reports revealed that &agr;-smooth muscle actin–expressing myofibroblasts are primarily responsible for fibrosis. It is believed that myofibroblasts are differentiated from resident fibroblasts, whereas the transformation of vascular endothelial cells into myofibroblasts, known as endothelial-mesenchymal transition, has been suggested to be intimately associated with perivascular fibrosis. Thus, we hypothesized that HGF prevents cardiac fibrosis by blocking these pathways. We analyzed the pressure-overloaded HGF-transgenic mouse model made by transverse aortic constriction. Human coronary artery endothelial cells and human cardiac fibroblasts were examined in vitro after being treated with transforming growth factor-&bgr;1 or angiotensin II with or without HGF. The amount of cardiac fibrosis significantly decreased in pressure-overloaded HGF-transgenic mice compared with pressure-overloaded nontransgenic controls, particularly in the perivascular region. This was accompanied by a reduction in the expression levels of fibrosis-related genes and by significant preservation of echocardiographic measurements of cardiac function in the HGF-transgenic mice (P<0.05). The survival rate 2 months after transverse aortic constriction was higher by 45% (P<0.05). HGF inhibited the differentiation of human coronary artery endothelial cells into myofibroblasts induced by transforming growth factor-&bgr;1 and the phenotypic conversion of human cardiac fibroblasts into myofibroblasts. We conclude that HGF reduced cardiac fibrosis by inhibiting endothelial-mesenchymal transition and the transformation of fibroblasts into myofibroblasts.