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Severe pulmonary vascular disease, such as pulmonary hypertension (PH) which is characterized by proliferation of pulmonary artery smooth muscle cells and progression of pulmonary arterial fibrosis, is not curable without lung transplantation. Thus further development of alternative therapies is required. This article, using rat and piglet model of advanced pulmonary vascular diseases, shows that gene transfer with Hepatocyte Growth Factor (HGF) inhibited overgrowth of pulmonary artery smooth muscle cells and reduced lung expression level of endothelin-1 and TGF-beta, which prevented vascular medial hyperplasia and matrix accumulation of pulmonary arteries, as well as induced angiogenesis. HGF gene transfer also enhanced the ameliorating effect of PGIS in experimental PH. These results suggest that gene transfer with HGF may be found suitable in treating subjects with severe pulmonary vascular disease.

Regenerative medicine using growth factors is an important therapeutic tool in patients with tissue ischemia or defects. Among such therapies, we have mainly addressed the sustained release of basic fibroblast growth factor (bFGF) as a safe and effective treatment. We developed a biodegradable hydrogel composed of acidic gelatin to enable bFGF to be released at the site of action for a sufficient time period. We experimentally applied this method in various animal models for cardiovascular research with satisfactory results as follows: 1) Sustained release of bFGF from gelatin hydrogel is effective in ischemic heart disease, dilated cardiomyopathy, pulmonary hypertension, limb ischemia, and bone regeneration; 2) Both sustained release of bFGF and bone marrow cell transplantation induce angiogenesis, but only bFGF produces arteriogenesis; 3) bFGF increases efficacy of cell transplantation, such as cardiomyocyte or skeletal myoblast. Future study is warranted for safe and effective clinical applications of the bFGF controlled-release system.

Cardiac regeneration therapy was recently attempted for recovering heart failure. However it is not clear whether the therapy is effective for severe failing hearts that need mechanical circulatory assistance. We examined a gene therapy for angiogenesis after acute myocardial infarction in goats under ventricular assist system (VAS).

Six adult goats (56–65kg) were created with impaired hearts by ligating the coronary artery and installing pulsatile bi-ventricular assist devices (VADs). Hepatocyte Growth Factor (HGF) was selected as a gene for an angiogenesis factor, which also has cardio-protective activities. The HGF group (n=3) were administered 2.0mg human HGF-cDNA plasmid in myocardium. The control group (n=3) were similarly administered beta-galactosidase plasmid. Four weeks after gene transfection, we tried to wean all goats from VADs.

The myocardia transfected with the hHGF-cDNA contained hHGF protein at levels as high as 1.0+/−0.3ng/g tissue 3 days after transfection. After weaning from VADs, the HGF group showed good hemodynamics, while the control group showed deterioration. The percent fractional shortening was significantly higher in the HGF group than the control group (HGF vs. control, 37.9+/−1.7% vs. 26.4+/−0.3%, p<0.01). LV dilatation associated with myocytes hypertrophy and fibrotic changes were detected in the control group, but not in the HGF group. Vascular density was markedly increased in the HGF group.

These results suggest that gene therapy using hHGF may enhance the chance of “bridge to recovery” in the impaired heart under VAS.

Therapeutic delivery of bone marrow derived mononuclear cells (BMMNCs) into ischemic tissues would be able to improve blood flow. Vascular endothelial growth factor (VEGF) is known as a potent angiogenic growth factor. We injected sFLT-1 expressing plasmid into mice anterotibial muscle. One week after gene transfer, gap unilateral femoral arterial segments were removed and murine BMMNCs (1×10 cells) were delivered into the thigh muscle to make BMMNC delivery model. SFLT-1 gene transfer inhibited BMMNCs delivery-induced blood flow recovery. This finding suggests that the effect of BMMNCs delivery is at least partially mediated via VEGF.

Background: Therapeutic angiogenesis by using cells is being performed in Japan. However, it is unknown in how many centers and how these therapies are performed. The efficacy and side effects are also unknown. Thus, we conducted the survey by mailing questionnaire within Japan. Methods and results: Two surveys were performed in 2003. The first survey unveiled that cell therapy was performed in 32 facilities until October 2003. The second survey unveiled the followings. (1) The total number of performed cases was 221. 153 patients (69.2%) had arterio-sclerosis obliterance (ASO), 56 patients (25.3%) thromboangitis obliterans (TAO, Burger’s disease), and 12 patients (5.4%) other conditions. (2) The sources of cells were bone marrow-mononuclear cells (61.5%), peripheral-mononuclear cells (9.5%), and peripheral CD34 cells (22.1%). A few patients (6.7%) were treated with a cytokine only (granulocyte-colony stimulating factor: G-CSF). (3) Inclusion criteria were the same for most facilities, such as patients with PAOD, especially with critical limb ischemia with rest pain, non-healing ischemic ulcers and non-candidates for non-surgical or surgical revascularization. All facilities excluded patients with histories of malignant disorder during the past 5 years, proliferative diabetic retinopathy, pregnancy, proliferative blood disease, uncontrolled ischemic heart disease, rheumatic arthritis, or psychiatric disease. (4) Subjective improvement was observed in 138 of 199 patients (69%). Objective improvements for ABI, TcO2 or angiographic findings were observed in 98 of 182 patients (53.8%). (6) Three of 221 patients (1.4%) died after cell therapy. One died from cerebro-vascular attack (thrombo-embolizm), and two died from acute myocardial infarction (AMI). Conclusion: Our clinical survey has shown that cell therapy is being performed in many medical centers in Japan. It seems to be safe and effective for patients with PAOD with no surgical options

Conventional Anigiographic Findings in Autologous Bone Marrow Mononuclear Cells Transplantation for Critical Limb Ischemia: Bone marrow mononuclear cells have many of the characteristics of stem cells for mesenchymal tissues, and secrete many angiogenic cytokines. We performed autologous transplantation of bone marrow mononuclear cells in six patients with critical limb ischemia due to Buerger disease, who were not candidates for catheter or surgical revascularization. Leg pains at rest and skin ulcers improved after bone marrow transplantation in all patients, although significant collateral developments after the therapy by conventional angiography could not be observed. Autologous transplantation of bone marrow mononuclear cells including stem cells improved critical limb ischemia due to Buerger disease. Neovascularization after therapeutic angiogenesis might be quite small and could not be visualized by conventional angiography.

Novel Micro-angiograpy: We developed in-hospital micro-angiographic equipment which consisted of a high power X-ray source for computed tomography and an avalanche type detector characterized by a high spatial resolution (20 µm) and high sensitivity (100 times of CCD camera). We visualized mid-zone collaterals after femoral arterial exfoliation with and without therapeutic angiogenesis in rabbit ischemic limbs and assessed the radio-absorptions in a clinical setting. The micro-angiography clearly demonstrated mid-zone collaterals after the treatment with a diameter of down to 50µm, but the conventional angiography did not. The sum of radio-absorptions for 10 seconds in clinical settings was 300 mSv. The newly developed in-house micro-angiography could illuminate micro-vessels with a diameter of down to 50µm in clinical settings safely and could be useful in the evaluation of therapeutic angiogenesis.

Therapeutic angiogenesis has been successfully induced by various methods, and developed clinically as a new treatment for ischemic diseases. We have tried to induce therapeutic angiogenesis for the treatment of ischemic heart disease by the implantation of bone marrow mononuclear cells (BM-MNCs), because BM-MNCs consist of complex populations, including the endothelial progenitor and angiogenic cytokine-producing cells. Using the ischemic heart model in rats and dogs, we have demonstrated that the intramyocardial implantation of BM-MNCs is a feasible and safe method for inducing therapeutic angiogenesis and improving deteriorated cardiac function. Then, we performed the intramyocardial implantation of autologous BM-MNCs in eight patients with ischemic heart disease who underwent coronary arterial bypass graft (CABG) simultaneously. A specific increase in myocardial perfusion was seen in five patients, and two patients showed improved left ventricular wall motion, 1 and 12 months after treatment. No postoperative complications, new-onset malignant arrhythmias, mass formation or calcification, or any other abnormal signs related with this new therapy were detected in any of the patients by 3 years follow-up examination. Although further investigation on the safety and effect of cell-based therapy are required, our preliminary results indicated that the intramyocardial injection of BM-MNCs is a feasible and safe method of inducing therapeutic angiogenesis and improving cardiac function in patients with severe ischemic heart disease.

Background: Endothelial progenitor cells in the CD34 stem-cell fraction of adult human peripheral blood take a part in postnatal neovascularization after mobilization from bone marrow. We investigated whether transplantation of bone marrow-mononuclear cells (BM-MNCs), including endothelial progenitor cells (EPCs), into ischemic limbs rescues ischemia in patients with peripheral artery occulusive disease (PAOD). Methods and Results: 22 patients with severe PAOD (atherosclerosis obliterance and Thromboangiitis obliterance / Burger’s disease) were enrolled. After collecting MNCs from the autologous bone marrow using the density gradient centrifugation method, we transplanted them into the ischemic limb. Four weeks after transplantation, pain quantified by the Visual Analog Scale was significantly improved. Skin ulcers healed in 6 of 10 patients and the size was reduced in the rest of them. Ankle brachial blood pressure ratio index (ABI) increased (0.58 ±0.2 to 0.69±0.26 at 4weeks). Furthermore, most of patients revealed angiographical improvements. No adverse event was observed.

Conclusion: Our findings indicate that therapeutic angiogenesis by transplantation of autologous BM-MNCs is effective and safe for patients with PAOD.

A 60 year-old man with atherosclerosis obliterans had received a bypass operation twice, but came to notice resting pain in the left calf. As it was not possible to perform further bypass operations (resulys of arteriography), we conducted conservative treatments. We therefore performed therapeutic angiogenesis by autologous transplantation of bone marrow mononuclear cells since the symptoms were not improved at all. Resting pain was improved the next day after the operation and completely disappeared on the third day. The patient was only able to walk 180m in the preoperative state by crutch-assisted, but walked 180m without a stick on the second day and 900m on the fourth day. This treatment did not cause any inconvenience in everyday life and the patient showed no resting pain eight weeks later. In addition, the ankle-brachial index improved to 0.48 from the preoperation level of 0.36 after two months.

Therapeutic angiogenesis by autologous transplantation of bone marrow mononuclear cells was very useful.

Critical limb ischemia in Buerger’s disease may lead to limb loss because reconstructive surgery is usually impossible. Recently, the autologous bone-marrow implantation into an ischemic limb has been demonstrated to enhance angiogenesis. We applied this new modality to two patients in Fontaine IV. The two patients were successfully treated without major amputations and we did not recognize any complications related to the bone-marrow implantation. Autologous bone-marrow implantation was effective for critical limb ischemia of Buerger’s disease by promoting neoangiogenesis. However, the long-term efficacy of this treatment may be limited, probably because it does not control the vasculitis.