Natsuo Inoue

Mechanisms Underlying the Impairment of Ischemia-Induced Neovascularization in Matrix Metalloproteinase 2–Deficient Mice

Matrix metalloproteinases (MMPs) have been implicated in the process of neovascularization. However, the exact roles of individual MMPs in vessel formation are poorly understood. To study the putative role of MMP-2 in ischemia-induced neovascularization, a hindlimb ischemia model was applied to MMP-2+/+ and MMP-2−/− mice. Serial laser Doppler blood-flow analysis revealed that the recovery of the ischemic/normal blood-flow ratio in MMP-2−/− young and old mice remained impaired throughout the follow-up period. At day 35, microangiography and anti–l-lectin immunohistochemical staining revealed lesser developed collateral vessels and capillary formation in both old and young MMP-2−/− mice compared with the age-matched MMP-2+/+ mice. An aortic-ring culture assay showed a markedly impaired angiogenic response in MMP-2−/− mice, which was partially recovered by supplementation of the culture medium with recombinant MMP-2. Aorta-derived endothelial cells or bone marrow–derived endothelial progenitor cell (EPC)-like c-Kit+ cells from MMP-2−/− showed marked impairment of invasive or/and proliferative abilities. At day 7, plasma and ischemic tissues of vascular endothelial growth factor protein were reduced in MMP-2−/−. Flow cytometry showed that the numbers of EPC-like CD31+c-Kit+ cells in peripheral blood markedly decreased in MMP-2–deficient mice. Transplantation of bone marrow–derived mononuclear cells from MMP-2+/+ mice restored neovascularization in MMP-2−/− young mice. These data suggest that MMP-2 deficiency impairs ischemia-induced neovascularization through a reduction of endothelial cell and EPC invasive and/or proliferative activities and EPC mobilization.

Role of Bradykinin, Nitric Oxide, and Angiotensin II Type 2 Receptor in Imidapril-Induced Angiogenesis

The angiotensin II (Ang II)-Ang II type 1 receptor pathway is proangiogenic, whereas studies showed that some angiotensin-converting enzyme inhibitors also stimulate angiogenesis in the setting of tissue ischemia, leaving a controversy of Ang II-mediated angiogenesis. We investigated whether an angiotensin-converting enzyme inhibitor imidapril-induced angiogenesis might be mediated via the tissue bradykinin pathway. To rule out the conventional effects of Ang II on angiogenesis, we used Ang II type 1a receptor knockout (AT1aKO) mice. We examined the effects of the angiotensin-converting enzyme inhibitor imidapril on angiogenesis in a hindlimb ischemia model using AT1aKO mice. After induction of hindlimb ischemia, AT1aKO mice were treated with or without imidapril (1.0 or 0.1 mg/kg per day for 21 days). Angiogenesis was quantified by laser Doppler blood flowmetry and capillary density. Angiogenesis was reduced in AT1aKO mice compared with wild-type mice. Imidapril with either low or high doses enhanced angiogenesis in AT1aKO mice (P<0.01). Ang II type 2 receptor antagonist (PD123319; 30 mg/kg per day) and B1 receptor antagonist (DesArg9-[Leu8]-bradykinin; 50 nmol/kg per day) suppressed the imidapril-induced angiogenesis in AT1aKO mice to an extent even lower than that of nontreated AT1aKO mice. B2 receptor antagonist (Hoechst 140; 100 &mgr;g/kg/d) and NO synthase inhibitor (NG-nitro-l-arginine methyl ester; 20 mg/kg per day) moderately attenuated the imidapril-mediated angiogenesis. RT-PCR revealed that vascular endothelial growth factor receptor 2 mRNA was reduced with PD123319, DesArg9-[Leu8]-bradykinin, or Hoechst 140, and vascular endothelial growth factor mRNA abundance was suppressed with PD123319 or DesArg9-[Leu8]-bradykinin. In conclusion, imidapril elicited angiogenesis in the setting of tissue ischemia in AT1aKO mice. This angiogenic effect might involve the Ang II-Ang II type 2 receptor pathway in addition to the bradykinin-B1 and bradykinin-B2 receptor/NO-dependent pathways.

Combination of In Vivo Angiopoietin-1 Gene Transfer and Autologous Bone Marrow Cell Implantation for Functional Therapeutic Angiogenesis

Objective—Autologous bone marrow mononuclear cell (BM-MNC) implantation into ischemic tissues promotes angiogenesis, but a large amount of marrow aspiration is required, which is a major clinical limitation. Angiopoietin-1 (Ang-1) is requisite for vascular maturation during angiogenesis. We examined the impacts of combinatorial Ang-1 gene transfer and low-dose autologous BM-MNC implantation on therapeutic angiogenesis in a rabbit model of hind limb ischemia. Methods and Results—Rabbits were divided into 4 groups: phosphate-buffered saline (control), 500 &mgr;g Ang-1 plasmid (Ang-1), 1×106 autologous BM-MNCs (BMC), and Ang-1 plasmid plus BM-MNCs (combination). The Ang-1 group had a greater angiographic score and capillary density compared with the control (P<0.05), but the Ang-1 gene therapy alone did not improve transcutaneous oxygen pressure (TcO2) and skin ulcer score. However, the combination group showed a significant improvement in not only angiographic score and capillary density (P<0.05) but also TcO2 (P<0.05) and skin ulcer score. These efficacies were greater in the combination group compared with the BMC group. Conclusions—This Ang-1 gene and BM-MNC combination therapy enhances not only quantitative but also qualitative angiogenesis in ischemic tissues. Moreover, the combination therapy will enable a reduction in the amount of BM aspiration required for significant therapeutic angiogenesis.

Therapeutic Angiogenesis Using Novel Vascular Endothelial Growth Factor-E/Human Placental Growth Factor Chimera Genes

Background—Vascular endothelial growth factor-A (VEGF-A) promotes angiogenesis but causes adverse side effects such as edema or tissue inflammation. VEGF-E, found in the genome of the Orf virus, specifically binds to VEGF receptor-2 and shows mitotic activity on endothelial cells. Recently, we created two forms of VEGF-E and human placental growth factor (PlGF) chimera genes (VEGF-E chimera #9 and VEGF-E chimera #33), which are humanized genes with VEGF-E function but showing less antigenicity. Methods and Results—We examined potential proangiogenic activities of these chimera genes. Four types of expression plasmids (pCDNA3.1-LacZ, phVEGF-A, pVEGF-Echimera#9, and pVEGF-Echimera#33) were administered in a rat model of hindlimb ischemia. Either pVEGF-Echimera#9, pVEGF-Echimera#33, or phVEGF-A significantly increased the ratio of ischemic/normal hindlimb blood-flow compared with the control pCDNA3.1-LacZ treated group (by 1.5-fold, 1.5-fold, and 1.4-fold, respectively, P<0.05). Histochemical staining by alkaline phosphatase also revealed that either pVEGF-Echimera#9, pVEGF-Echimera#33, or phVEGF-A increased the capillary density compared with the pCDNA3.1-LacZ treated group (1.4-fold, 1.5-fold, and 1.5-fold, respectively, P<0.05). Furthermore, immunostaining for anti-ED1 revealed that fewer macrophages had infiltrated in both pVEGF-Echimera#9 and pVEGF-Echimera#33 groups compared with the phVEGF-A group (P<0.05). Conclusions—Novel VEGF-E/human PlGF chimera genes, pVEGF-Echimera#9, and pVEGF-Echimera#33 significantly stimulated angiogenesis in response to tissue ischemia to an almost identical extent to that induced by phVEGF-A with fewer tissue inflammation responses.

The incidence and clinical significance of non-isolation of the pulmonary vein carina after encircling ipsilateral pulmonary veins isolation for paroxysmal atrial fibrillation: a pitfall of the double-Lasso technique

AIMS Encircling ipsilateral pulmonary veins (PVs) isolation (EIPVsI) with the double-Lasso technique has proven to be effective to cure atrial fibrillation (AF). However, in this technique, PV mapping with circular catheters may miss a non-isolation of the PV carina. The purpose of this study was to reveal the incidence and clinical significance of a non-isolation of the PV carina after EIPVsI. METHODS AND RESULTS We studied 81 consecutive paroxysmal AF patients (age 61 ± 12 years, 56 men), in whom EIPVsI was successfully performed in one encircling line with the endpoint of the demonstration of bidirectional conduction block between the PVs and left atrium (LA) with the double-Lasso technique. After a successful EIPVsI, pacing from the PV carina was performed and it captured the LA in 17 (21.0%) patients. During a mean follow-up period of 19 ± 13 months, AF recurred in 13 (16.0%) patients. A multivariate Cox proportional analysis revealed that a non-isolation of the PV carina after the EIPVsI was a significant predictor (hazard ratio = 3.91, 95% confidence interval = 1.13-14.16, P = 0.03) of AF recurrence. CONCLUSIONS Pulmonary vein mapping with the double-Lasso technique did miss the non-isolation of the PV carina after a successful EIPVsI, which was an independent predictor of AF recurrence after the EIPVsI. Pacing from the PV carina may be required to confirm the electrical isolation of the PV carina after EIPVsI with the double-Lasso technique.

Renin–angiotensin system inhibitors can suppress atrial fibrillation recurrence after encircling ipsilateral pulmonary vein isolation in patients with a non-dilated left atrium

Introduction: The purpose of this study was to investigate whether the effects of renin–angiotensin system inhibitors (RASIs) after encircling ipsilateral pulmonary veins isolation (EIPVsI) for atrial fibrillation (AF) differed between patients with non-dilated and dilated left atria. Materials and methods: We retrospectively studied 292 consecutive patients (mean age=61±11 years, 75% males) who underwent successful EIPVsI for paroxysmal or persistent AF. RASIs’ effects were compared between the patients with a non-dilated left atrium of <40 mm (n=178) and dilated left atrium of ≥40 mm (n=114). Results: During a mean follow-up period of 18.9±12.7 months, AF recurred in 38 (21.4%) and 45 (39.5%) patients with non-dilated and dilated left atria, respectively. A multivariate Cox proportional analysis revealed that treatment with RASIs (hazard ratio (HR) 0.30, 95% confidence interval (CI) =0.13–0.66, p=0.003), the duration of AF (HR 1.08/year, 95% CI=1.01–1.16, p=0.03), a history of hypertension (HR 2.86, 95% CI=1.21–6.85, p=0.02) and the left ventricular ejection fraction (HR 0.54/10%↑, 95% CI=0.34–0.87, p=0.01) were associated with AF recurrences in patients with a non-dilated left atrium. On the other hand, only the duration of AF (HR 1.11/year, 95% CI=1.01–1.21, p=0.03) was associated with AF recurrences in those with a dilated LA, and RASIs had no effect on AF recurrences (p=0.65). Conclusions: RASIs suppressed AF recurrences after EIPVsI only in patients with a non-dilated left atrium.

THE PREVALENCE AND CLINICAL SIGNIFICANCE OF CARINA-LA CONDUCTION AFTER SUCCESSFUL PULMONARY VEIN ISOLATION FOR ATRIAL FIBRILLATION

Background: Bidirectional block between pulmonary veins (PVs) and left atrium (LA) is the gold standard to confirm the successful procedure of pulmonary vein isolation (PVI) for atrial fibrillation (AF). However, conduction between carina and LA remains in some cases after PVI, and its clinical significance has remained unclear. The purpose of this study is to clarify the incidence and clinical significance of carina-LA conduction after PVI.