The transplantation of cardiomyocytes has been shown to be an attractive and useful strategy for cardiac functional improvement after myocardial damage by Yokomura and colleagues at Toho University. This group also demonstrated that cardiomyocytes can be cryopreserved.

In the present study, they evaluated whether transplanted cryopreserved rat cardiomyocytes would survive in the connective tissue of the hind limb in the adult rat.

Cryopreserved samples (using a freezing medium to reach minus 80 degrees Celsius, then stored in liquid nitrogen for 1, 2, 4, 8, 12 and 24 weeks) were shown on histology after thawing to look quite similar to non-cryopreserved cells, with no morphological differences (Figure 1). Figure 2 shows microscopic images of cryopreserved and non-cryopreserved cardiomyocytes. No differences were observed in cell proliferation at each time point, and cell proliferation gradually decreased at the same rate in the cryopreserved and fresh cardiomyocytes over time. No difference was observed in the percentage of cryopreserved beating cells compared to fresh cells at each time point, and the percentage gradually decreased in both groups over time.

An in vivo study was then conducted using the same methodology, except the cells were cryopreserved for one week. Cardiomyocytes were injected using a tuberculin syringe into the subcutaneous tissue of the adult rat. Cyclosporin A (5 mg/kg) was administered subcutaneously daily. The rats were sacrificed at 4 weeks after transplantation. Survival and contractility of the transplanted tissue were evaluated visually, histologically, and by electrocardiogram. The electrical activity of the recipient heart was about 219 beats per minute (bpm), while the electrical activity of the cardiac-like tissue formed from transplanted cryopreserved cardiomyocytes was 60 bpm. On histology, the non-cryopreserved tissue size was smaller and infiltration of lymphocytes was similar to that of cryopreserved cells. New blood vessel-like tissue was observed in the transplant region.

The function and morphology of tissue derived from cryopreserved cardiomyocytes and the cultured cardiomyocytes were similar to that of non-cryopreserved cells, stated Hiroki Yokomuro, MD. Transplanted cryopreserved cardiomyoctyes survived and contracted spontaneously in connective tissue. Cryopreservation likely affected the reduction of immunogenecity for transplanted cells. The investigators concluded that this storage technique may be applied to future cell transplantation.

Gene transfer by viral vectors is an efficient but biohazardous process. Naked DNA transfer, although safer, is less efficient. Mori and colleagues demonstrated that angiogenic gene therapy can be potentiated using biodegradeable gelatin hydrogel (GHG). A non-viral, but highly efficient gene therapy for salvaging ischemic rabbit hindlimb was achieved with fibroblast growth factor-4 (FGF4)-GHG (FGF4-GHG). The degree of development and maturity of the therapeutic angiogenesis were evidenced by synchrotron radiation (SR) microangiography.

In 6 transfected rabbits, FGF4 transgene expression was evaluated on day 17 of hindlimb ischemia. RT-PCR analysis revealed FGF4 transgene expression in all injection sites with naked FGF4 and FGF4-GHG. However, expression was detected 10 mm apart from the injection site in the FGF4-GHG-treated animal, but not in the naked FGF4-treated animal.

Then one of three gene therapies was performed 10 days after femoral artery resection: 1) LacZ 500mcg + GHG (n=8), 2) naked FGF4 500 mcg (n=7), and 3) FGF4 500 mcg + GHG (n=7). On day 38, conventional angiography, SR microangiography, gross anatomical and molecular biological studies were performed.

Severe toe necrosis and thigh muscle atrophy and necrosis were noted in the control rabbit treated with LacZ-GHG. In contrast, ischemic tissue damage was nearly negligible in FGF4-GHG-treated rabbits. Severe tissue damage was noted in the LacZ-GHG rabbit, less damage in the naked FGF4 rabbit, and the least tissue damage in the FGF4-GHG rabbits. All of the differences were statistically significant.

Conventional angiography showed that in the naked FGF4 and FGF4-GHG-treated rabbits that the midzone collaterals developed substantially. However, no statistical difference was seen between the groups. SR microangiography showed that flow reserve is preserved in the FGF4-GHG rabbit, while flow still phenomenon developed in the naked FGF4 rabbits, demonstrating a greater degree of angiogenesis in the FGF4-GHG rabbits. A statistically significant difference between the groups was shown by the angiographic score ratio.

In a subsequent study using the same protocol in the canine model, these investigators demonstrated significantly enhanced fractional shortening and higher flow reserve in the FGF4-GHG-treated animals, compared to LacZ-treated animals.

Bone marrow (BM) is a natural source of endothelial progenitor cells (EPC) and a broad spectrum of cytokines. Bone marrow-mononuclear cells (BM-MNC) containing EPC are mobilized from BM in response to tissue ischemia or VEGF therapy to accumulate in ischemic lesions.

Autologous implantation of BM-MNC markedly augmented new capillary formation in ischemic myocardium, resulting in increased regional blood flow and improvement in cardiac function in a study conducted by Kamihata and colleagues at Kansai Medical University. BM-MNC synthesized and secreted angiogenic ligands such as basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF) and angiopoiten-1. BM-MNC implanted in ischemic myocardium was incorporated into capillary vessel walls, stated Kamihata.

BM-MNC derived from mini-swine ileum were injected into the ischemic zone. A control group was injected with medium alone into the ischemic zone after irradiation. Angiogenesis was evaluated at week 3. The myocardium was removed for assessment of the infarcted area and imunohistochemistry.

The distal portion of the LAD was visible in all animals on coronary angiography. However, the number of visible collateral vessels branching from the left circumflex coronary artery in the direction of the infarct tended to increase in the BM-MNC-implanted animals compared to the control animals.

A perfusion defect was seen at week 3 in the control animals on MCE using second Harmonic technology, which was significantly different from baseline, and a persistent flow deficit in the ischemic region. In the BM-MNC-implanted animals, the perfusion defect was markedly reduced, as much as 83% compared to the baseline value.

The LVEF was significantly improved by 48% in the BM-MNC-implanted group at 3 weeks after irradiation, while it was decreased by 11% in the control animals from baseline values. The extent of the maximum left ventricle DPTT deterioration was less in the BM-MNC-implanted group compared to the control group.

The number of capillaries in the ischemic portion increased about 2.5-3.0-fold in the BMI group, compared to control as seen on immunohistochemistry. Analysis revealed that 34% of vessels incorporated BM-MNC. Since not all cells incorporated MNC, the investigators hypothesized that the MNC may release angiogenic factors in transplanted myocardium to enhance angiogenesis. On further examination, they found that MNC expressed more mRNA for bFGF than for VEGF and angiopoietin-1, but not greater than angiopoieten-2.

Autologous bone marrow implantation may constitute a novel a strategy for achieving optimal therapeutic angiogenesis, concluded Kamihata, by exploiting the natural ability of bone marrow cells to secrete important angiogenic factors and its ability to incorporate into foci of neo-vascularization.

Intramyocardial transfection of genes encoding growth factors such as VEGF constitutes an alternative strategy for patients with severe myocardial ischemia. Two clinical reports have shown the beneficial effect of VEGF DNA for therapeutic angiogenesis. Hepatocyte growth factor (HGF) has been postulated as a potential growth factor, with potential cell protective functions and angiogenesis, apoptosis, and anti-fibrosis properties with a specific HFG receptor (c-Met). However, the role of HGF in the ischemic heart, especially its role in angiogenesis, has not been clarified.

Sawa and colleagues at Osaka University have reported that HGF and c-Met is upregulated in ischemic myocardium in rat hearts, and is limited to the ischemic region. Gene transfection of HGF prevents ischemia and reperfusion injury in rat hearts. This evidence supports the notion that the HGF/c-Met system may play a role in the angiogenesis of ischemic myocardium. In the present study, the effect of direct injection of plasmid DNA of human HGF as therapeutic angiogenesis was investigated using the canine heart. Left coronary angiography revealed that the LAD was supplied with collateral vessels enhanced by HGF injection. The capillary count in the ischemic myocardium increased significantly after direct injection of HGF. Regional contractile function and blood flow in the ischemic areas showed significant recovery after HGF treatment. No significant difference in leukocyte number was observed between the groups.

Twenty-two canines divided into 3 groups were given HGF-cDNA (125 mcg injection; Group H), LacZ cDNA (125 mcg injection; Group L) and a sham control group (Group S). Ligation was performed at the mid-portion of the LAD, just below the first diagonal branch. After1 month of LAD ligation, direct injection was performed at six ischemic points.

At 4 days after gene transfection, Group H showed marked expression of human FGF, while Groups L and S did not. One month after gene transfection the LAD, undetectable before transfection, was supplied by neo-vasculature, i.e., the angiogenic effect of FGF gene transfection was visible. The number of factor VIII positive endothelial cells was markedly increased in Group H compared to Group L on histology. The capillary density in the ischemic myocardium was significantly higher in Group H than in the two other groups. The percentage of thickening fraction was significantly higher and the regional blood flow in ischemic myocardium was significantly improved in Group H than in the other two groups.

VEGF has a potent angiogenic function but it increases membrane permeability. HGF has angiogenic function and increases proliferation of endothelial cells, but not smooth muscle cells. Therefore, these investigators compared the effect of HGF and VEGF plasmid transfection using the canine LAD ligation model. The number of factor VIII positive endothelial cells was markedly increased in both groups, compared to the LacZ group. The density of capillaries in the ischemic myocardium was significantly higher in the HGF and VEGF groups compared to LacZ and sham groups. Only the VEGF group showed a higher value in terms of water content, suggesting increased membrane permeability in this group.

The direct injection of plasmid DNA encoding human HGF into the ischemic myocardium may be an angiogenic therapy improving regional perfusion and contractile function, concluded Yoshiki Sawa, MD. Thus, continuous local production of HGF may be considered as a novel therapeutic angiogenesis strategy for ischemic heart disease, such as myocardial infarction. Based on these data, they are preparing a protocol for a clinical trial using HGF.