Background: Periostin (POSTN) is a 93 kDa matrix protein that helps to regulate collagen gene expression in the extracellular matrix. POSTN overexpression is a prognostic factor in malignant cancers; however, some researchers have observed it in the stroma, whereas others have reported it on tumors. Objective: This study aimed to investigate the function of POSTN on tumors. Methods and Results: We found that POSTN in cancer cells can be detected by using an antibody against the POSTN C-terminal region exon 17 (Ex17 antibody), but not with an antibody against the POSTN N-terminal region exon 12 (Ex12 antibody) in patients with breast cancer. In a fraction secreted from fibroblasts, LC–MS/MS analysis revealed a short fragment of POSTN of approximately 40 kDa with exon 17. In addition, molecular interaction analysis showed that POSTN with exon 17, but not POSTN without exon 17, bound specifically to wnt3a, and the Ex17 antibody inhibited the binding. Conclusion: A short fragment of POSTN with exon 17, which originates in the fibroblasts, is transported to cancer cells, whereas POSTN fragments without exon 17 are retained in the stroma. The Ex17 antibody inhibits the binding between POSTN exon 17 and wnt3a.

Objective— Peripheral arterial disease is highly prevalent in the elderly and in the subjects with cardiovascular risk factors such as diabetes. Approximately 2% to 4% of those affected with peripheral arterial disease commonly complain of intermittent claudication. Cilostazol, a type III phosphodiesterase inhibitor, is the only Food and Drug Administration–approved drug for the treatment of intermittent claudication. Cilostazol has been shown to be beneficial for the improvement of pain-free walking distance in patients with intermittent claudication in a series of randomized clinical trials. However, the underlying mechanism how cilostazol improved intermittent claudication symptoms is still unclear. Approach and Results— In this study, the effect of cilostazol on ischemic leg was investigated in mouse ischemic hindlimb model. Administration of cilostazol significantly increased the expression of hepatocyte growth factor (HGF), vascular endothelial growth factor, angiopoietin-1, and peroxisome proliferator-activated receptor-γ in vasculature. The capillary density in ischemic leg was also significantly increased in cilostazol treatment group when compared with control and aspirin treatment group. However, an increase in capillary density and the expression of growth factors was almost completely abolished by coadministration of HGF-neutralizing antibody, suggesting that cilostazol enhanced angiogenesis mainly through HGF. In vitro experiment revealed that cilostazol treatment increased HGF production in vascular smooth muscle cells via 2 major pathways: peroxisome proliferator-activated receptor-γ and cAMP pathways. Conclusions— Our data suggest that the favorable effects of cilostazol on ischemic leg might be through the angiogenesis through the induction of HGF via peroxisome proliferator-activated receptor-γ and cAMP pathways.

Objective: Peripheral arterial disease (PAD) is highly prevalent in the elderly as well as in the subjects with cardiovascular risk factors such as diabetes. Approximately 2–4% of those affected with PAD commonly complain of intermittent claudication (IC). Cilostazol, a type III phosphodiesterase inhibitor, is the only FDA-approved drug for the treatment of IC. Cilostazol has been shown to be beneficial for the improvement of pain-free walking distance in patients with IC in a series of randomized clinical trials. However, the underlying mechanism how cilostazol improved IC symptoms is still unclear. Design and Method: In this study, the effect of cilostazol on ischemic leg was investigated in mouse ischemic hind limb model. Results: Administration of cilostazol significantly increased the expression of hepatocyte growth factor (HGF), vascular endothelial growth factor, angiopoietin-1, and peroxisome proliferator-activated receptor (PPAR)-γ in vasculature. The capillary density in ischemic leg was also significantly increased in cilostazol treatment group as compared to control and aspirin treatment group. However, an increase in capillary density and the expression of growth factors was almost completely abolished by co-administration of HGF-neutralizing antibody, suggesting that cilostazol enhanced angiogenesis mainly through HGF. In vitro experiment revealed that cilostazol treatment increased HGF production in vascular smooth muscle cells via two major pathways, PPAR-γ and cAMP pathways. Conclusions: Our data suggest that the favorable effects of cilostazol on ischemic leg might be through the angiogenesis through the induction of HGF via PPAR-γ and cAMP pathways.

Objective: Although mortality rate due to acute myocardial infarction (AMI) has been extremely improved by the development of early reperfusion therapy, the sudden death due to acute heart failure, lethal arrhythmias, and chronic heart failure post AMI are still clinically important issue. We previously reported that periostin expression significantly increased mainly in myofibroblast following AMI and over-expression of full length of periostin (Pn variant 1) resulted in ventricular dilation with enhanced interstitial collagen deposition in rat model. However, other reports documented that short form splicing variants, Pn variant 2 (lacking exon-17) or Pn variant 4 (lacking exon-17 and -21), promoted cardiac repair by angiogenesis or preventing cardiac rupture following AMI. Design and Method: Therefore, we employed neutralizing antibody (PnAb) that selectively inhibit Pn variant 1 by blockade of exon-17 and tested in rat AMI model. Results: Administration of PnAb resulted in a significant decrease in infarcted and fibrotic area of myocardium and preserved ejection fraction and preventing wall thinning and elevation of LVEDP at 2 months. Strikingly, PnAb significantly increased residual myocytes area in ischemic region. Moreover, the level of Pn 1 expression at day 7 was correlated significantly with the severity of AMI. In vitro study demonstrated that Pn 1, but not Pn 2 or 4, inhibited fibroblast and myocyte attachment and caused myocyte apoptosis, which might account for cell slippage observed during cardiac remodeling. Conclusions: In summary, Pn variant 1 expression significantly increased during cardiac remodeling, and inhibited anchorage- dependent myocyte growth and survival. Specific neutralizing antibody against Pn exon-17, Pn variant 1, without suppressing other Pn variants significantly suppressed cardiac remodeling post AMI and might offer a new class of medication for the treatment of AMI patients.

We previously reported that overexpression of full-length periostin, Pn-1, resulted in ventricular dilation with enhanced interstitial collagen deposition in a rat model. However, other reports have documented that the short-form splice variants Pn-2 (lacking exon 17) and Pn-4 (lacking exons 17 and 21) promoted cardiac repair by angiogenesis and prevented cardiac rupture after acute myocardial infarction. The apparently differing findings from those reports prompted us to use a neutralizing antibody to selectively inhibit Pn-1 by blockade of exon 17 in a rat acute myocardial infarction model. Administration of Pn neutralizing antibody resulted in a significant decrease in the infarcted and fibrotic areas of the myocardium, which prevented ventricular wall thinning and dilatation. The inhibition of fibrosis by Pn neutralizing antibody was associated with a significant decrease in gene expression of fibrotic markers, including collagen I, collagen III, and transforming growth factor-β1. Importantly, the number of α-smooth muscle actin–positive myofibroblasts was significantly reduced in the hearts of animals treated with Pn neutralizing antibody, whereas cardiomyocyte proliferation and angiogenesis were comparable in the IgG and neutralizing antibody groups. Moreover, the level of Pn-1 expression was significantly correlated with the severity of myocardial infarction. In addition, Pn-1, but not Pn-2 or Pn-4, inhibited fibroblast and myocyte attachment, which might account for the cell slippage observed during cardiac remodeling. Collectively, these results indicate that therapeutics that specifically inhibit Pn exon-17, via a neutralizing antibody or drug, without suppressing other perisotin variants might offer a new class of medication for the treatment of acute myocardial infarction patients.

Peripheral arterial disease (PAD) is highly prevalent in the elderly and subjects with atherosclerotic risk factors. Approximately 2-4% of those affected with PAD commonly complain of intermittent claudication (IC) that is a strong indicator for the development of systemic atherosclerosis. Cilostazol (CSZ) has been shown to be of benefit in improving pain-free walking distance and quality of life in patients with IC. CSZ may be the most clinically effective pharmacologic option for IC. However, the underlying mechanism for amelioration of IC by CSZ remains largely unclear. Here, we demonstrate that CSZ increases expression of hepatocyte growth factor (HGF) in vascular smooth muscle cells (SMCs) via two distinct pathways, PPAR γ and cAMP pathway. Activation of PPAR γ and cAMP activates HGF promoter leading to increase in capillary density and improvement of perfusion in mouse ischemic hind limb model. Enhancement of angiogenesis by CSZ involves Akt/eNOS activation in endothelial cells by HGF secreted from SMCs. However, this process was essentially absent in aspirin treatment group and CSZ with HGF neutralizing antibody treatment group. As CSZ also increases HGF protein production in interstitial area of skeletal muscle where satellite cells reside, we postulated that CSZ stimulates myogenesis. To proof this hypothesis, BrdU was injected every day for 7days in mouse hind limb model, and animals were sacrificed at day 7 and 28. Embryonic form of myocyte heavy chain (eMHC), BrdU positive myocyte, and nuclear centralization was measured. As predicted, CSZ increases the number of newly formed myocyte in ischemic leg compared to control and Aspirin treated mouse. This beneficial effect of CSZ was blunted by the administration of HGF neutralizing antibody. Additionally, Heart specific HGF overexpression mouse in which serum level of HGF is 4-5 times higher than wild type mouse were generated to proof the role of HGF in angiogenesis and myogenesis. Similar to the data obtained from CSZ treated mouse, HGF Tg mouse ameliorates ischemic hind limb via both angiogenesis and myogenesis. Altogether, our date demonstrates that CSZ improves ischemic hind limb via angiogenesis and myogenesis. This results may account for the beneficial effect of CSZ in PAD patients with IC.

Introduction: Despite the remarkable progress of medicine and endovascular procedures for revascularization, patients with critical limb ischemia (CLI) remain at high risk for amputation and often have a low quality of life due to pain and ulcers in the ischemic leg. Thus, a novel strategy for generating new blood vessels in CLI patients without treatment options is vital. Pre-clinical studies and Phase I clinical trials using VEGF and fibroblast growth factor (FGF) demonstrated promising results; however, more rigorous Phase II and III clinical trials failed to demonstrate benefits for CLI patients. Recently, two multicenter, double-blind, placebo-controlled clinical trials in Japan (Phase III) and the USA (Phase II) showed the benefits of hepatocyte growth factor (HGF) gene therapy for CLI patients. Although the number of patients included in these trials was relatively small, these results imply a distinct beneficial function for HGF over other angiogenic growth factors in a clinical setting.Areas covered: In this review, data from Phase I–III clinical trials of gene therapy for patients with peripheral artery disease (PAD) are examined. In addition, the potential mechanisms behind the success or failure of clinical trials are discussed.Expert opinion: Compared with VEGF and FGF, HGF has a unique molecular effect on inflammation, fibrosis and cell senescence under pathological conditions. These features may explain the clinical benefits of HGF in PAD patients.