Nira Varda-Bloom

Intracoronary injection of basic fibroblast growth factor enhances angiogenesis in infarcted swine myocardium.

OBJECTIVES This study was performed to examine the effect of intracoronary exogenous basic fibroblast growth factor (bFGF) on angiogenesis in infarcted myocardial regions. BACKGROUND Exogenous bFGF is a potent promoter of angiogenesis. Little information is available on its effect on myocardial angiogenesis. METHODS Myocardial infarction was induced in 10 pigs by intracoronary injection of microscopic beads. Four pigs served as a control group; in six pigs slow-release bFGF was delivered by the beads. Cardiac performance was evaluated by repeated echocardiographic measurement and angiogenesis was evaluated by immunohistochemical studies 14 days later. RESULTS As compared with control pigs, pigs treated with bFGF had higher microvessel counts (mean +/- SEM) in both viable tissue (141 +/- 27 per field vs. 39 +/- 4, p = 0.01) and nonviable tissue (329 +/- 26 per field vs. 95 +/- 7, p < 0.001) within the infarct area. No significant differences in total regional left ventricular wall motion were noted between the two groups throughout the 14-day study period. CONCLUSIONS In the swine, direct intracoronary application of bFGF to infarcted myocardium enhances myocardial neovascularization within 2 weeks.

Tissue-specific gene therapy directed to tumor angiogenesis

Gene therapy directed specifically to the vascular wall, particularly to angiogenic endothelial cells is a prerequisite in vascular disease treatment. Angiogenesis is a major feature in many pathological conditions including wound healing, solid tumors, developing metastases, ischemic heart diseases and diabetic retinopathy. In the present study we developed a tissue-specific gene therapy to the angiogenic blood vessels of tumor metastasis using an adeno-based vector containing the murine preproendothelin-1 (PPE-1) promoter. Genes activated by the PPE-1 promoter were highly expressed in bovine aortic endothelial cells in vitro. Systemic injection of the adenoviral vectors AdPPE-1-luciferase and AdCMV-luciferase to normal C57BL/6 mice, resulted in higher activity of PPE-1 promoter compared with CMV promoter in the aorta and vascularized tissues such as heart, kidney, lung and pancreas. Systemic administration of the adenoviral vector, in mice bearing Lewis lung carcinoma, resulted in high and specific activity of PPE-1 in the new vasculature of primary tumors and lung metastasis. Cellular distribution of the delivered gene revealed highest expression of GFP in angiogenic endothelial cells of the metastasis. We expect that this approach of ‘vascular-directed’ gene therapy will be applicable to both vascular diseases and cancer.

A Closed‐Chest Pig Model of Sustained Ventricular Tachycardia

The goal of this study was to develop and explore a closed‐chest animal model of sustained VT. Seven of 11 domestic pigs had successful induction of myocardiai infarction by injection of agarose gel microbeads into the left anterior descending coronary artery tbrougb an inflated balloon angioplasty catheter. Four of the first five pigs died and seem to represent a “learning experience.” During a 3‐ to 50‐day follow‐up period, each pig underwent 1 ‐3 electropbysiological studies. Sustained, monomorpbic VT was induced 1–4 times in 5 of the 7 pigs (a total of 19 episodes), was reproducible during the same study in all pigs, and could be repetitively induced during successive studies in some. Ventricular fibrillation was induced less frequently (nine episodes) and was successfully terminated by DC shock in eight episodes. We conclude tbat a closed‐cbest pig model of VT is feasible and is associated witb a relatively bigb induction rate of sustained, monomorpbic, and reproducible VT and a relatively low mortality rate.

Endothelial-targeted gene transfer of hypoxia-inducible factor-1α augments ischemic neovascularization following systemic administration

Hypoxia-inducible factor-1α (HIF-1α) is a key regulator of the response to low oxygen levels and has been used for therapeutic angiogenesis. Various routes of administration have been used for delivering genes to the ischemic region including the intramuscular (IM) and intraarterial routes. When compared with these delivery methods, the intravenous (IV) route confers many advantages, including less invasiveness and lower cost. However, its use is hampered by the fact that it does not result in specific and robust tissue expression of the genes. Our aim was to determine the feasibility, safety, and therapeutic efficacy of systemic administration of adenoviral-mediated HIF-1α targeted to the endothelium. Using confocal microscopy and biodistribution studies we demonstrated that a modified murine preproendothelin-1 promoter (PPE1-3x) can target gene expression specifically to endothelial cells within ischemic muscle following systemic IV administration in C57BL/6 mice. Accordingly, an adenovirus expressing a PPE1-3x-regulated stabilized HIF-1α molecule, further activated by constitutive activation of its C-transactivation domain (C-TAD), was created. Systemic tail-vein administration of this adenovirus in a mouse hindlimb ischemia model resulted in enhanced blood perfusion, improved clinical outcome, and increased capillary density without systemic toxicity, in contrast to the profound systemic side effects and lack of therapeutic efficacy following cytomegalovirus (CMV)-regulated HIF-1α administration. Collectively, these data suggest that transcriptionally controlled systemic proangiogenic gene therapy is feasible, safe, and efficacious.Hypoxia-inducible factor-1alpha (HIF-1alpha) is a key regulator of the response to low oxygen levels and has been used for therapeutic angiogenesis. Various routes of administration have been used for delivering genes to the ischemic region including the intramuscular (IM) and intraarterial routes. When compared with these delivery methods, the intravenous (IV) route confers many advantages, including less invasiveness and lower cost. However, its use is hampered by the fact that it does not result in specific and robust tissue expression of the genes. Our aim was to determine the feasibility, safety, and therapeutic efficacy of systemic administration of adenoviral-mediated HIF-1alpha targeted to the endothelium. Using confocal microscopy and biodistribution studies we demonstrated that a modified murine preproendothelin-1 promoter (PPE1-3x) can target gene expression specifically to endothelial cells within ischemic muscle following systemic IV administration in C57BL/6 mice. Accordingly, an adenovirus expressing a PPE1-3x-regulated stabilized HIF-1alpha molecule, further activated by constitutive activation of its C-transactivation domain (C-TAD), was created. Systemic tail-vein administration of this adenovirus in a mouse hindlimb ischemia model resulted in enhanced blood perfusion, improved clinical outcome, and increased capillary density without systemic toxicity, in contrast to the profound systemic side effects and lack of therapeutic efficacy following cytomegalovirus (CMV)-regulated HIF-1alpha administration. Collectively, these data suggest that transcriptionally controlled systemic proangiogenic gene therapy is feasible, safe, and efficacious.

Phase 1/2 study of nilotinib prophylaxis after allogeneic stem cell transplantation in patients with advanced chronic myeloid leukemia or philadelphia chromosome-positive acute lymphoblastic leukemia

Allogeneic stem cell transplantation (SCT) remains the standard treatment for advanced chronic myeloid leukemia (CML) and Philadelphia chromosome–positive acute lymphoblastic leukemia (Ph+ALL). Relapsed disease is the major cause of treatment failure, especially when SCT is given in the setting of advanced disease. Tyrosine kinase inhibitors can be given after transplantation prophylactically or after the detection of minimal residual disease (MRD) to reduce the relapse risk.

Vascular Wall Maturation and Prolonged Angiogenic Effect by Endothelial-Specific Platelet-Derived Growth Factor Expression

Background: The implementation of angiogenic gene therapy at clinics is hindered by the transience of the therapeutic effect. Recruiting vascular wall smooth muscle cells, a process termed ‘maturation’, can stabilize newly formed vessels. Objective: To induce angiogenesis followed by vessel maturation in a murine ischemic limb model by endothelial cell-specific promoter regulated expression of vascular endothelial growth factor (VEGF) and platelet-derived growth factor-BB (PDGF-BB). Methods: We constructed adenoviral vectors containing angiogenic factors VEGF and PDGF-B regulated by a modified preproendothelin-1 (PPE-1-3x) promoter and investigated their angiogenic effect in a murine ischemic limb model. Results: VEGF gene therapy increased perfusion and the vessel density in the limb shortly after expression with PPE-1-3x promoter or cytomegalovirus (CMV) promoter vectors, but only PPE-1-3xVEGF treatment exhibited a sustained effect. Expression of PDGF-B by PPE-1-3x promoter resulted in morphological maturation of the vasculature and further increased the perfusion, while nonspecific expression of PDGF-B with CMV promoter had no therapeutic effect. Regulation of dual therapy with VEGF and PDGF-B by PPE-1-3x promoter resulted in an early-onset, sustained angiogenic effect accompanied by vessel maturation. Conclusions: Systemic gene therapy with the angiogenic factors VEGF and PDGF-B under angiogenic- endothelial cell-specific regulation was effective in inducing functionally and morphologically mature vasculature.

Specific induction of tumor neovasculature death by modified murine PPE-1 promoter armed with HSV-TK.

Background: One strategy to increase tissue specificity of gene therapy is to use promoters or enhancers. Objectives: (1) To enhance the selectivity of a murine preproendothelin-1 (PPE-1) promoter in tumor angiogenesis by using a positive endothelial transcription-binding element. (2) To test the specificity and efficiency of the modified PPE-1 promoter [PPE-1(3X)] in vitro and in vivo by using reporter genes, and the therapeutic gene herpes simplex virus-thymidine kinase (HSV-TK) in a mouse model of Lewis lung carcinoma (LLC). Results: The modified PPE-1 promoter specifically induced expression in the tumor angiogenic vascular bed with a 35-fold higher expression compared to the normal vasculare bed of the lung. Thus, when the HSV-TK gene controlled by the modified PPE-1 promoter was used systemically, it induced tumor-specific necrosis, apoptosis and mononuclear infiltrates, leading to massive destruction of the neovasculature of the pulmonary metastasis, which suppressed metastasis development. Conclusions: These results show that an adenoviral vector armed with HSV-TK controlled by the endothelial-selective murine PPE-1(3X) promoter is efficient and safe to target tumor neovasculature.

Systemic administration of radiation-potentiated anti-angiogenic gene therapy against primary and metastatic cancer based on transcriptionally controlled HSV-TK.

Transcription-targeted gene delivery directed against angiogenic endothelial cells is a new approach against advanced cancer. Moreover, the herpes simplex virus-thymidine kinase (HSV-TK) gene coupled with low dose radiotherapy is an efficient and externally controlled cytotoxic system. We have previously demonstrated enhanced endothelial-specific cell expression and killing using the modified murine pre-proendothelin-1 promoter (PPE1-3x) to direct adenoviral expression of a pro-apoptotic gene. The purpose of this study was to create an externally potentiated systemic antiangiogenic gene delivery system based on an adenoviral vector expressing HSV-TK under the regulation of PPE1-3X promoter combined with radiotherapy for eradicating metastatic cancer. Ad-PPE1-3x-TK induced endothelial-specific cell killing in-vitro upon introduction of the prodrug ganciclovir (GCV). BALB/c mice bearing a primary CT-26 colon carcinoma tumor showed tumor growth suppression and diminished tumor angiogenesis when the vector was administered intravenously, activated with GCV and potentiated with a single sub-therapeutic and non-toxic radiation dose. Moreover, intravenous administration of the vector, activated with GCV and potentiated with chest aimed radiation, to C57BL/6 mice bearing Lewis lung carcinoma metastases resulted in prolongation of mice survival. PPE1-3x-regulated HSV-TK expression was detected only in lung metastases in contrast to CMV-regulated expression. This novel system may benefit patients with metastatic disease.

Noninvasive evaluation of microcirculatory hemodynamic changes during hemorrhage followed by saline or blood transfusion.

ABSTRACT The purpose of the study was to examine the ability of a system combining laser Doppler flowmetry (LDF), photoplethysmograph (PPG), and transcutaneous oxygen tension (tc-PO2) to follow changes in the microcirculation during hemorrhage and following blood or saline return, and to test the hypothesis that such changes precede and might predict changes in the systemic blood pressure. Measurements were performed on the skin of anesthetized rabbits (n = 10) during mild (0–8%), moderate (9–24%), and severe (25–30% of blood volume) hemorrhage, and following complete volume restitution by blood or saline. We found the following: 1) hemorrhage caused typical changes in the LDF, PPG, and tc-PO2 signals that could be formulated by mathematical models, 2) these signals identified blood as being more efficient than saline for volume restitution following hemorrhage, and 3) microcirculatory changes precede and might predict systemic hemodynamic events.

Electrical injury in the femoral artery of rabbits as a model for arterial thrombosis: a pilot study.

Electrical stimulation was delivered to the femoral artery of 20 rabbits to examine whether endothelial injury results in a consistent formation of arterial thrombosis. The arterial patency was monitored throughout the experiment by flowmeter and was visu alized by femoral angiography in 5/20 cases. The arterial segments remained totally occluded in 7/20 (35%), partially occluded in 9/20 (45%), and patent in 4/20 (20%) rabbits following stimulation with 200 μA anodal current for 180 minutes. The average time of electrical stimulation needed to achieve total occlusion (n = 7) was 110 ±49 minutes. Alternating occlusion and recanalization of the artery (cyclic flow variation) was observed in 12 rabbits, with total occlusion ensuing in 6/12 cases. Intravenous administration of recombinant tissue-type plasminogen activator (20 μg/kg/min for sixty minutes) resulted in femoral reflow and subsequent reocclusion in 2/5 cases. Histopathologic examination disclosed arterial thrombi composed of platelets, fibrin, and red blood cells. Thus, according to these data this technique was found to induce arterial thrombosis following electrical stimulation of the rabbit femoral artery but was inconsistent regarding the arterial patency.