Shin-ichiro Hayashi

Therapeutic Angiogenesis Induced by Human Recombinant Hepatocyte Growth Factor in Rabbit Hind Limb Ischemia Model as Cytokine Supplement Therapy

Hepatocyte growth factor (HGF) exclusively stimulates the growth of endothelial cells without replication of vascular smooth muscle cells and acts as a survival factor against endothelial cell death. Therefore we hypothesized that a decrease in local vascular HGF might be related to the pathogenesis of peripheral arterial disease. We initially evaluated vascular HGF concentration in the vessels of patients with arteriosclerosis obliterans. Consistent with in vitro findings that hypoxia downregulated vascular HGF production, vascular HGF concentration in the diseased segments of vessels from patients with arteriosclerosis obliterans was significantly decreased as compared with disease-free segments from the same patients (P<0.05), accompanied by a marked reduction in HGF mRNA. On the other hand, a novel therapeutic strategy for ischemic diseases that uses angiogenic growth factors to expedite and/or augment collateral artery development has recently been proposed. Thus in view of the decreased endogenous vascular HGF, rhHGF (500 micrograms/animal) was intra-arterially administered through the internal iliac artery of rabbits in which the femoral artery was excised to induce unilateral hind limb ischemia, to evaluate the angiogenic activity of HGF, which could potentially have a beneficial effect in hypoxia. Administration of rhHGF twice on days 10 and 12 after surgery produced significant augmentation of collateral vessel development on day 30 in the ischemic model as assessed by angiography (P<0.01). Serial angiograms revealed progressive linear extension of collateral arteries from the origin stem artery to the distal point of the reconstituted parent vessel in HGF-treated animals. In addition, we examined the feasibility of intravenous administration of rhHGF in a moderate ischemia model. Importantly, intravenous administration of rhHGF also resulted in a significant increase in angiographic score as compared with vehicle (P<0.01). Overall, a decrease in vascular HGF might be related to the pathogenesis of peripheral arterial disease. In the presence of decreased endogenous HGF, administration of rhHGF induced therapeutic angiogenesis in the rabbit ischemic hind limb model, as potential cytokine supplement therapy for peripheral arterial disease.

Hepatocyte growth factor is a novel member of the endothelium-specific growth factors: additive stimulatory effect of hepatocyte growth factor with basic fibroblast growth factor but not with vascular endothelial growth factor.

Objective To seek an endothelium-specific growth factor by examining the mitogenic effects of hepatocyte growth factor (HGF) on endothelial cells and on vascular smooth muscle cells (VSMC). Methods Rat and human endothelial cells and VSMC were employed. DNA, RNA and protein synthesis were measured by using [3H]-thymidine, uridine and leucine. Coculture of endothelial cells with VSMC was also performed to study the role of endothelial cells. Results Coculture of endothelial cells with VSMC resulted in a significant decrease in DNA synthesis of VSMC. HGF, as well as basic fibroblast growth factor (bFGF), stimulated DNA, RNA and protein synthesis by endothelial cells in a dose-dependent manner. Interestingly, co-incubation of endothelial cells with HGF and bFGF resulted in an additive stimulation of DNA synthesis. Similarly, HGF and interleukin-1 α and -6 stimulated DNA synthesis by coronary endothelial cells, whereas interleukin-1 β and transforming growth factor-β (TGF-β) did not. However, HGF showed markedly different actions from bFGF on VSMC growth. bFGF, TGF-β, interleukin-1α, -1β and -6 stimulated DNA synthesis in VSMC significantly, whereas HGF did not. Finally, we examined the mitogenic effect of HGF on human aortic endothelial cells and VSMC. Incubation with HGF increased DNA synthesis and growth by endothelial cells in a dosedependent manner, whose degree was significantly greater than those with bFGF, vascular endothelial growth factor (VEGF) and interleukin-6. Addition of HGF and VEGF showed no additive effect on DNA synthesis in endothelial cells, in contrast to those of bFGF and HGF. On the other hand, bFGF, but not HGF and VEGF, stimulated DNA synthesis in VSMC. Conclusion These results demonstrate that HGF can exert stimulating effects on endothelial cell growth, but not on VSMC growth, in an additive manner with bFGF but not with VEGF. These characteristics of HGF as an endotheliumspecific growth factor may provide the opportunity for a new therapeutic strategy for vascular diseases in which the abnormalities are vasoconstriction and pathological growth.

Estrogen-Mediated Endothelial Progenitor Cell Biology and Kinetics For Physiological Postnatal Vasculogenesis

Estrogen has been demonstrated to promote therapeutic reendothelialization after vascular injury by bone marrow (BM)–derived endothelial progenitor cell (EPC) mobilization and phenotypic modulation. We investigated the primary hypothesis that estrogen regulates physiological postnatal vasculogenesis by modulating bioactivity of BM-derived EPCs through the estrogen receptor (ER), in cyclic hormonally regulated endometrial neovascularization. Cultured human EPCs from peripheral blood mononuclear cells (PB-MNCs) disclosed consistent gene expression of ER &agr; as well as downregulated gene expressions of ER &bgr;. Under the physiological concentrations of estrogen (17&bgr;-estradiol, E2), proliferation and migration were stimulated, whereas apoptosis was inhibited on day 7 cultured EPCs. These estrogen-induced activities were blocked by the receptor antagonist, ICI182,780 (ICI). In BM transplanted (BMT) mice with ovariectomy (OVX) from transgenic mice overexpressing &bgr;-galactosidase (lacZ) regulated by an endothelial specific Tie-2 promoter (Tie-2/lacZ/BM), the uterus demonstrated a significant increase in BM-derived EPCs (lacZ expressing cells) incorporated into neovasculatures detected by CD31 immunohistochemistry after E2 administration. The BM-derived EPCs that were incorporated into the uterus dominantly expressed ER &agr;, rather than ER &bgr; in BMT mice from BM of transgenic mice overexpressing EGFP regulated by Tie-2 promoter with OVX (Tie-2/EGFP/BMT/OVX) by ERs fluorescence immunohistochemistry. An in vitro assay for colony forming activity as well as flow cytometry for CD133, CD34, KDR, and VE-cadherin, using human PB-MNCs at 5 stages of the female menstrual-cycle (early-proliferative, pre-ovulatory, post-ovulatory, mid-luteal, late-luteal), revealed cycle-specific regulation of EPC kinetics. These findings demonstrate that physiological postnatal vasculogenesis involves cyclic, E2-regulated bioactivity of BM-derived EPCs, predominantly through the ER&agr;.

Cyclic AMP Inhibited Proliferation of Human Aortic Vascular Smooth Muscle Cells, Accompanied by Induction of p53 and p21

Although cAMP is an important second messenger that plays a pivotal role in the regulation of platelet aggregation and dilatation of blood vessels, little is known about the action of cAMP on the growth of vascular smooth muscle cells (VSMCs). Thus, we initially studied the effects of cAMP accumulation by using various cAMP stimulants, including a phosphodiesterase type 3 inhibitor (cilostazol) on human aortic VSMC growth. Accumulation of cAMP inhibited the platelet-derived growth factor (PDGF)-stimulated VSMC growth in a dose-dependent manner (P<0.01), whereas PDGF significantly stimulated the growth of human VSMCs. Thus, we focused on the role of cell cycle regulatory genes, especially on a negative regulator, an anti-oncogene, p53. The protein of p53 was potentiated by cilostazol as well as forskolin and 8-bromo-cAMP, whereas PDGF decreased p53 expression. Upregulation of p53 protein by cAMP was further confirmed by the observation that the decrease in p21, a p53-inducible protein, by PDGF was significantly attenuated by cilostazol in a dose-dependent manner (P<0.01). These results revealed that accumulation of cAMP inhibited VSMC proliferation, which was at least in part due to an increase in p53-p21 expression. Because p53 and p21 have been reported to induce apoptosis, we examined apoptotic cells for cAMP accumulation. Incubation of VSMCs with cilostazol resulted in a significant increase in apoptotic cells in a dose-dependent manner compared with vehicle treatment as assessed by nuclear chromatic morphology (P<0.01); forskolin also stimulated apoptotic cells. Consistent with nuclear staining, DNA fragmentation in VSMCs treated with forskolin as well as 8-bromo-cAMP and cilostazol was significantly increased compared with DNA fragmentation in VSMCs treated with vehicle, whereas PDGF significantly decreased the rate of DNA fragmentation (P<0.01). Overall, these results demonstrated that cAMP inhibited the proliferation of human aortic VSMCs, accompanied by p53-p21-mediated apoptosis. Analogues of cAMP that have direct inhibitory effects on VSMC proliferation can be considered as potential antiproliferative drugs against VSMC growth.

Negative Regulation of Local Hepatocyte Growth Factor Expression by Angiotensin II and Transforming Growth Factor-β in Blood Vessels: Potential Role of HGF in Cardiovascular Disease

Because hepatocyte growth factor (HGF) is a member of the endothelium-specific growth factors, we hypothesized that HGF may play a role in cardiovascular disease. Therefore we first examined the role of local HGF production in endothelial cell (EC) growth. Addition of anti-HGF antibody to EC resulted in a significant decrease in EC number. Moreover, coculture of vascular smooth muscle cells (VSMC) with EC resulted in an increase in EC number that was completely inhibited by anti-HGF antibody, suggesting that HGF secreted from EC and VSMC regulates EC growth in an autocrine-paracrine manner. Interestingly, transforming growth factor (TGF)-ss significantly decreased HGF secretion from EC, whereas interleukin 6 stimulated immunoreactive HGF secretion. In human VSMC, TGF-ss and angiotensin II suppressed local HGF production in a dose-dependent manner. Interestingly, anti-TGF-beta antibody resulted in significant but not complete inhibition of the decrease in local HGF production. To further study the regulation of local HGF production, we used a coculture system. Coculture of VSMC with EC resulted in a significant decrease in local HGF secretion. The decrease in local HGF production by coculture was significantly attenuated by anti-TGF-beta antibody, suggesting that inhibition of local HGF production in the coculture system was due to TGF-beta activation. Moreover, a further decrease in local HGF production in the coculture system by angiotensin II was also observed. Finally, we studied the role of angiotensin II in the regulation of the local HGF system in vivo by using a balloon injury rat model. Of importance, local HGF production was significantly decreased in balloon-injured arteries compared with intact vessels, accompanied by a reduction of HGF mRNA. An angiotensin-converting enzyme inhibitor (cilazapril) or an angiotensin II type 1 receptor antagonist (E-4177) significantly stimulated local vascular HGF production associated with the inhibition of neointimal formation after balloon injury compared with vehicle. In contrast, hydralazine did not alter local HGF production or neointimal formation despite decreasing blood pressure to a similar level as that in rats treated with cilazapril or E-4177. Overall, local HGF secretion from vascular cells was negatively regulated by TGF-beta and angiotensin II. The present study also demonstrated that blockade of angiotensin II significantly inhibited neointimal formation, accompanied by a significant increase in local vascular HGF production in vivo in the balloon injury model. Given the strong mitogenic activity of HGF on endothelial cells, increased local HGF production by blockade of angiotensin II may enhance reendothelialization after balloon injury. Downregulation of the local vascular HGF system by TGF-beta and vascular angiotensin may play an important role in the pathogenesis of cardiovascular diseases.

Contribution of Bcl-2, but Not Bcl-xL and Bax, to Antiapoptotic Actions of Hepatocyte Growth Factor in Hypoxia-Conditioned Human Endothelial Cells

Abstract—Angiogenic growth factors play important roles in angiogenic responses, such as vasculogenesis and angiogenesis in response to hypoxia. A novel angiogenic growth factor, hepatocyte growth factor (HGF), has been reported to inhibit endothelial cell death. However, its molecular mechanisms are largely unknown. Thus, we studied (1) the effects of HGF on hypoxia-induced endothelial apoptosis and (2) the molecular mechanisms of the antiapoptotic actions of HGF in endothelial cells. Severe hypoxia increased the cell death rate in human aortic endothelial cells, whereas HGF significantly attenuated cell death. In addition, hypoxic treatment resulted in a significant increase in apoptotic cells, whereas HGF could attenuate apoptosis, accompanied by attenuation of the increase in caspase-3–like activity (P <0.01). Of importance, HGF significantly increased Bcl-2, an inhibitor of apoptosis, in a dose-dependent manner under normoxic and hypoxic conditions (P <0.01), whereas hypoxic conditions resulted in a significant decrease in Bcl-2. In contrast, HGF failed to affect Bcl-xL, which is also well known as an inhibitor of apoptosis under both normoxic and hypoxic conditions, whereas Bcl-xL was significantly decreased in endothelial cells exposed to hypoxia (P <0.01). No significant change in Bax, a promoter of apoptosis, was also observed in endothelial cells under hypoxia, whereas HGF did not affect Bax. Overall, this study demonstrated that HGF prevented endothelial cell death induced by hypoxia through its antiapoptotic action. The antiapoptotic mechanisms of HGF in hypoxia-induced endothelial cell death largely depend on Bcl-2, but not Bcl-xL and Bax.

Inhibition of the p53 tumor suppressor gene results in growth of human aortic vascular smooth muscle cells. Potential role of p53 in regulation of vascular smooth muscle cell growth.

Loss of activity of the p53 tumor suppressor gene product has been postulated in the pathogenesis of human restenosis. Although the antioncogenes p53 and retinoblastoma (Rb) susceptibility gene have been reported to play a pivotal role in cell cycle progression in various cells, the role of p53 and Rb in the growth of human vascular smooth muscle cells (VSMC) has not yet been clarified. We used antisense strategy against p53 and Rb genes by the viral envelope-liposomal method. Transfection of antisense p53 oligodeoxynucleotides (ODN) alone resulted in an increase in DNA synthesis compared with control (P<0.01). Similarly, transfection of antisense Rb ODN alone resulted in a higher DNA synthesis rate than control (P<0.01). Moreover, increase in VSMC number was only induced by transfection of antisense p53 ODN alone or cotransfection of p53/Rb ODN (P<0.01), whereas a single transfection of antisense Rb ODN had little effect on cell number. Therefore, we hypothesized that this discrepancy is due to the induction of apoptosis mediated by p53. Interestingly, apoptotic cells were markedly increased in VSMC transfected with antisense Rb ODN alone, accompanied by the induction of p53 protein. The number of apoptotic cells was attenuated by cotransfection of antisense p53 ODN (P<0.01). We finally examined the molecular mechanisms of apoptosis induced by the absence of Rb. In VSMC transfected with antisense Rb ODN, bax, a promoter of apoptosis, was significantly increased in VSMC transfected with antisense Rb ODN (P<0.01), whereas bcl-2 and Fas did not play a pivotal role in the induction of apoptosis. Overall, these data first demonstrated that the antioncogenes p53 and Rb negatively regulated the cell cycle in VSMC, suggesting that the modulation of their activity may mediate VSMC growth such as that in restenosis and atherosclerosis. The presence of p53 plays a pivotal role in the regulation of apoptosis in human VSMC growth, probably through the bax pathway. These results provide evidence that p53 is a functional link between cell growth and apoptosis in VSMC.

Inhibition of Growth of Human Vascular Smooth Muscle Cells by Overexpression of p21 Gene Through Induction of Apoptosis

The senescent cell-derived inhibitor (sdi)-1 protein (p21 product) has been identified as a downstream mediator of the tumor suppressor p53 in the regulation of cell cycle progression through a G1 phase checkpoint. Given the importance of cell cycle inhibition for the treatment of restenosis, in this study we focused on the function of p21 gene in inhibiting proliferation of vascular smooth muscle cells (VSMC). To test the hypothesis, we transfected human p21 gene into human aortic VSMC using hemagglutinating virus of Japan-liposome-mediated transfer. Initially, we examined the successful transfection of human p21 gene into VSMC. p21 protein was increased in VSMC transfected with p21 vector as compared with control vector. Accompanied by increased p21 protein, transfection of p21 vector resulted in a significant decrease in number of VSMC induced by 2% serum (P<.01). Although p21 has been reported to play an important role in the regulation of apoptosis in some cells, apoptosis mediated by p21 is still controversial. Therefore, we hypothesized that overexpression of p21 mediates apoptosis in human VSMC, in addition to the blockade of cell cycle progression. First, we assessed the concordance between morphologic analysis and apoptosis as determined by nuclear staining with Hoechst 33342. Cells transfected with p21 gene exhibited the characteristic features of cell shrinkage, membrane blebbing, and rounding that are typical of apoptotic death. Of greater interest, a significant increase in apoptotic cells was observed in VSMC transfected with p21 vector as compared with control vector (P<.01). These results were confirmed by the measurement of DNA fragmentation. Consistent with nuclear staining, DNA fragmentation in VSMC transfected with human p21 gene was significantly increased as compared with that in VSMC transfected with control vector (P<.05). To study the molecular mechanisms of apoptosis mediated by overexpression of p21 gene, the protein levels of bax, a promoter of apoptosis, and bcl-2, an inhibitor of apoptosis, were also measured by Western blotting. Overexpression of p21 gene significantly increased protein of bax (P<.05), whereas transfection of p21 gene did not alter bcl-2 protein. Importantly, the ratio of bax to bcl-2 was significantly increased in VSMC transfected with human p21 vector as compared with control vector (P<.05). Overall, these results demonstrated that inhibition of VSMC growth by overexpression of human p21 gene was accompanied by induction of apoptosis through an inappropriate increase in bax protein. These results suggest that regulation of cell cycle by p21 may be closely linked to programmed cell death/apoptosis in human VSMC.

Hepatocyte growth factor as a potential index of complication in diabetes mellitus

Objective Diabetes mellitus (DM), characterized by the premature development of microvascular and macrovascular disease, shows a loss of vasodilatory properties of resistance vessels. However, the mechanisms of endothelial dysfunction in diabetes have not yet been clarified. Hepatocyte growth factor (HGF) in vascular cells was down-regulated by high glucose levels, potentially accelerating the endothelial dysfunction in DM. In this study, the serum HGF level was measured to investigate further the role of HGF in DM. Methods Tissue and circulating HGF levels were measured in the KKAy mouse, a rodent model of non-insulin-dependent diabetes mellitus (NIDDM), and lean C57 BL control mice. Then, serum HGF concentrations were measured in NIDDM patients without liver, kidney or lung damage. For the study of serum HGF concentration, 30 normotensive and age-matched 58 DM patients were studied. The 58 DM patients were divided into 26 patients without hypertension and 32 patients with hypertension [22 patients without hypertensive complications (WHO I) and 10 patients with hypertensive complications (WHO II + III)]. Results The serum HGF concentration in KKAy mice was significantly lower than that in control mice (P < 0.01), at 14 weeks of age when they exhibit features of diabetes. Similarly, tissue HGF concentrations in the heart and kidney were decreased significantly in KKAy mice compared with control C57 BL mice (P < 0.05). The serum HGF concentration showed a significant negative correlation with hemoglobin (Hb) AIc concentration (P < 0.01, r = −0.41). Since the serum HGF concentration is a potential index of the severity of hypertension, the serum levels of HGF in DM patients without and with hypertension were examined. The serum HGF concentration in DM patients without hypertension was significantly lower than that in normal subjects (P < 0.05), whereas that in DM patients with hypertension was significantly higher than that in normal subjects (P < 0.01). Moreover, the serum HGF concentration in DM patients with hypertensive complications was further higher than that in others (P < 0.01). Conclusion The present data showed that serum, cardiac and renal HGF concentrations in KKAy mice were significantly decreased compared with control mice. Therefore, a decrease in local HGF may be a trigger of endothelial dysfunction in DM. Clinical data also demonstrated a significant negative correlation between serum HGF and HbAIc concentrations in diabetic patients without complications. In contrast, the serum concentration of HGF was significantly elevated depending on the severity of hypertension. These results suggest that HGF may be a new index of complications such as hypertension in DM.

Improvement of eosinophilic heart disease after steroid therapy: successful demonstration by endomyocardial biopsied specimens.

SummaryA 62-year-old man with acute eosinophilic endomyocarditis developed congestive heart failure. The biopsy specimens revealed degranulated eosinophils and eosinophil cationic protein (ECP) in the endocardium, and in activated eosinophils and the myocardial interstitium. On electronmicroscopy, a characteristic cardiac myocytolytic change showing disruption at the intercellular junctional site was observed. After steroid treatment, clinical symptoms, systolic dysfunction and laboratory data dramatically improved. In the subsequent biopsy specimens, eosinophilic infiltration and ECP disappeared. Steroid therapy provided a beneficial effect in preventing the progression of cardiac damage. This effect was clearly documented by histochemical studies of the serial endomyocardial biopsy samples.