Adenoviral VEGF-DΔN ΔC gene therapy for myocardial ischemia.

Background: Cardiovascular diseases are the leading cause of death globally. In spite of the availability of improved treatments, there is still a large group of chronic ischemia patients who suffer from significant symptoms and disability. Thus, there is a clear need to develop new treatment strategies for these patients. Therapeutic angiogenesis is a novel therapy method which has shown promising results in preclinical studies. In this study, we evaluated safety and efficacy of adenoviral (Ad) VEGF-DΔNΔC gene transfer for the treatment of myocardial ischemia in a pig model. Methods: Adenoviral VEGF-DΔNΔC gene transfer was given to pigs (n = 26) via intramyocardial injections using an electromechanical injection catheter. Angiogenic effects were evaluated in an acute myocardial infarction model (n = 18) and functionality of the lymphatic vessels were tested in healthy porcine myocardium (n = 8). AdLacZ was used as a control. Results: AdVEGF-DΔNΔC induced safe and effective myocardial angiogenesis by inducing a four-fold increase in mean capillary area at the edge of the myocardial infarct six days after the gene transfer relative to the control AdLacZ group. The effect was sustained over 21 days after the gene transfer, and there were no signs of vessels regression. AdVEGF-DΔNΔC also increased perfusion 3.4-fold near the infarct border zone relative to the control as measured by fluorescent microspheres. Ejection fraction was 8.7% higher in the AdVEGF-DΔNΔC treated group 21 days after the gene transfer relative to the AdLacZ control group. Modified Miles assay detected a transient increase in plasma protein extravasation after the AdVEGF-DΔNΔC treatment and a mild accumulation of pericardial effusate was observed at d6. However, AdVEGF-DΔNΔC also induced the growth of functional lymphatic vasculature, and the amount of pericardial fluid and level of vascular permeability had returned to normal by d21. Conclusion: Endovascular intramyocardial AdVEGF-DΔNΔC gene therapy proved to be safe and effective in the acute porcine myocardial infarction model and provides a new potential treatment option for patients with severe coronary heart disease.

The European Medicines Agency Review of Crizanlizumab for the Prevention of Recurrent Vaso-Occlusive Crises in Patients With Sickle Cell Disease.

Crizanlizumab is a monoclonal antibody that binds to P-selectin. On October 28, 2020, a conditional marketing authorization valid through the European Union (EU) was issued for crizanlizumab for the prevention of recurrent vaso-occlusive crises (VOCs) in patients with sickle cell disease aged 16 years or older. Crizanlizumab was evaluated in a phase 2, double-blind, placebo-controlled randomized multicenter trial comparing high-dose (5 mg/kg) crizanlizumab, low-dose (2.5 mg/kg) crizanlizumab and placebo in patients with a history of 2-10 VOCs in the previous year. Patients who were receiving concomitant hydroxycarbamide (HC) as well as those not receiving HC were included in the study. The primary endpoint of the trial was the annual rate of sickle cell-related pain crises as adjudicated by a central review committee. High-dose crizanlizumab led to a 45.3% lower median annual rate of sickle cell-related pain crises compared to placebo (P = 0.010), with no statistically significant difference for the low dose. Treatment with high-dose crizanlizumab led to similar incidences of adverse events (AEs), grade 3 AEs, and serious AEs compared to placebo. Most frequently observed AEs that occurred more often in the crizanlizumab arm compared to placebo were infusion related reactions (34.8% versus 21%), arthralgia (18.2% versus 8.1%), diarrhea (10.6% versus 3.2%), and nausea (18.2% versus 11.3%). The aim of this article is to summarize the scientific review of the application leading to regulatory approval in the EU.

EMA Review of Belantamab Mafodotin (Blenrep) for the Treatment of Adult Patients with Relapsed/Refractory Multiple Myeloma.

On August 25, 2020, a marketing authorization valid through the European Union was issued for belantamab mafodotin monotherapy for the treatment of multiple myeloma (MM) in adult patients who have received at least four prior therapies, whose disease is refractory to at least one proteasome inhibitor (PI), one immunomodulatory agent (IMiD), and an anti-CD38 monoclonal antibody (mAb), and who have demonstrated disease progression on the last therapy. Belantamab mafodotin is an antibody-drug conjugate that combines a mAb, which binds specifically to B-cell maturation antigen, with maleimidocaproyl monomethyl auristatin F, which is a cytotoxic agent. It was evaluated in Study 205678 (DREAMM-2), an open-label, two arm, phase II, multicenter study in patients with MM who had relapsed following treatment with at least three prior therapies, who were refractory to an IMiD, a PI, and an anti-CD38 mAb alone or in combination. Patients were randomized to receive 2.5 mg/kg (n = 97) or 3.4 mg/kg (n = 99) belantamab mafodotin by intravenous infusion every 3 weeks until disease progression or unacceptable toxicity. Belantamab mafodotin achieved an overall response rate (ORR) of 32% (97.5% confidence interval [CI]: 22-44) with a median duration of response (DoR) of 11 months (95% CI: 4.2 to not reached). The most frequently (≥20%) reported adverse reactions grades 3-4 with belantamab mafodotin were keratopathy (31%), thrombocytopenia (22%), and anemia (21%). With regard to the corneal risks associated with belantamab mafodotin, patients would need to undergo specific ophthalmic examinations so that any findings can be promptly and adequately managed. The scientific review concluded that a 32% ORR and a median DoR of 11 months observed with belantamab mafodotin was considered clinically meaningful. Given the manageable toxicity profile and considering that belantamab mafodotin has a mechanism of action that is different from that of authorized treatments in this group of highly pretreated patients whose disease is refractory to three classes of agents, the benefit risk for belantamab mafodotin monotherapy was considered positive, although the efficacy and safety evidence were not as comprehensive as normally required. IMPLICATIONS FOR PRACTICE: Belantamab mafodotin (Blenrep, GlaxoSmithKline, St. Louis, MO, U.S.A) was approved in the European Union as monotherapy for the treatment of adult patients with refractory/relapsed multiple myeloma. Belantamab mafodotin resulted in durable response in highly pretreated patients whose disease is refractory to three classes of agents. Belantamab mafodotin is a monoclonal antibody against B-cell maturation antigen conjugated with the potent antimitotic agent maleimidocaproyl monomethyl auristatin. This is the first monoclonal antibody to target this antigen in multiple myeloma, which represents a true novelty from a pharmacological point of view.

Susceptibility to Cardiac Arrhythmias and Sympathetic Nerve Growth in VEGF-B Overexpressing Myocardium.

VEGF-B gene therapy is a promising proangiogenic treatment for ischemic heart disease, but, unexpectedly, we found that high doses of VEGF-B promote ventricular arrhythmias (VAs). VEGF-B knockout, alpha myosin heavy-chain promoter (αMHC)-VEGF-B transgenic mice, and pigs transduced intramyocardially with adenoviral (Ad)VEGF- B186 were studied. Immunostaining showed a 2-fold increase in the number of nerves per field (76 vs. 39 in controls, p < 0.001) and an abnormal nerve distribution in the hypertrophic hearts of 11- to 20-month-old αMHC-VEGF-B mice. AdVEGF-B186 gene transfer (GT) led to local sprouting of nerve endings in pig myocardium (141 vs. 78 nerves per field in controls, p < 0.05). During dobutamine stress, 60% of the αMHC-VEGF-B hypertrophic mice had arrhythmias as compared to 7% in controls, and 20% of the AdVEGF-B186-transduced pigs and 100% of the combination of AdVEGF-B186- and AdsVEGFR-1-transduced pigs displayed VAs and even ventricular fibrillation. AdVEGF-B186 GT significantly increased the risk of sudden cardiac death in pigs when compared to any other GT with different VEGFs (hazard ratio, 500.5; 95% confidence interval [CI] 46.4-5,396.7; p < 0.0001). In gene expression analysis, VEGF-B induced the upregulation of Nr4a2, ATF6, and MANF in cardiomyocytes, molecules previously linked to nerve growth and differentiation. Thus, high AdVEGF-B186 overexpression induced nerve growth in the adult heart via a VEGFR-1 signaling-independent mechanism, leading to an increased risk of VA and sudden cardiac death.

Isolation of fresh endothelial cells from porcine heart for cardiovascular studies: a new fast protocol suitable for genomic, transcriptomic and cell biology studies.

BACKGROUND: Endothelial cells (ECs) play a key role in tissue homeostasis, in several pathological conditions, and specifically in the control of vascular functions. ECs are frequently used as in vitro model systems for cardiovascular studies and vascular biology. The porcine model is commonly used in human clinical cardiovascular studies. Currently, however, there is no robust protocol for the isolation of porcine heart ECs. We have developed a fast isolation protocol, which is cost effective, takes only 1-2 h, and produces EC purity of over 97%. This protocol is optimized for porcine hearts but can be adapted for use with other large animals. METHODS: Heart is washed by flushing with PBS, whereafter endothelial cells are detached by collagenase incubation and the cells can then be collected immediately after the incubation and plated within an hour after the heart is isolated from a pig. RESULTS: The swiftness of the protocol limits changes in the phenotype and RNA expression profile of the cells. Cells were identified as ECs with CD31 (PECAM-1) antibody immunostaining. Functionality of ECs were ensured with in vitro angiogenesis assay. The purity of the ECs was verified by using fluorescence assisted cell sorting (FACS) with the CD31 antibody. CONCLUSION: We developed a new, fast, and cost-effective isolation method for pig heart ECs. Successful isolation of pure ECs is a prerequisite for several cardiovascular and vascular biology studies.

Beyond endothelial cells: Vascular endothelial growth factors in heart, vascular anomalies and placenta.

Vascular endothelial growth factors regulate vascular and lymphatic growth. Dysregulation of VEGF signaling is connected to many pathological states, including hemangiomas, arteriovenous malformations and placental abnormalities. In heart, VEGF gene transfer induces myocardial angiogenesis. Besides vascular and lymphatic endothelial cells, VEGFs affect multiple other cell types. Understanding VEGF biology and its paracrine signaling properties will offer new targets for novel treatments of several diseases.

Advances and Challenges in Cardiovascular Gene Therapy.

Many promising cardiovascular gene therapy approaches have failed to fulfill expectations in clinical trials. However, 20 years of research and method development has laid a solid groundwork for future therapies, and the need for new treatment options still exists. The safety of gene therapy has been established with various viral vectors, transgenes and delivery methods. Improving success in clinical settings requires careful consideration of the translational process. This requires both improving animal models and preclinical end points, and new approach in patient recruitment and selection of clinical end points. This review focuses on bidirectional translationality from bench to bedside and back and proposes ways to improve the process. Developing a highly complex new therapy has taken an enormous amount of work and resources, but perhaps now after the hard lessons cardiovascular gene therapy is ready become a clinical reality.

AdVEGF-B186 and AdVEGF-DΔNΔC induce angiogenesis and increase perfusion in porcine myocardium.

OBJECTIVE: Coronary heart disease remains a significant clinical problem, and new therapies are needed especially for patients with refractory angina for whom the current therapies do not provide sufficient relief. The aim of this study was to find out if angiogenic gene therapy using new members of the vascular endothelial growth factor (VEGF) family, VEGF-B186 and VEGF-DΔNΔC, increase myocardial perfusion as measured by the positron emission tomography (PET) 15O-imaging, and whether there would be coronary steal effect to the contralateral side. Furthermore, safety of intramyocardial angiogenic adenoviral gene transfer was evaluated. METHODS: Intramyocardial adenoviral (Ad) VEGF-B186 or AdVEGF-DΔNΔC gene transfers were given endovascularly into the porcine posterolateral wall of the left ventricle (n=34). Six days later, PET 15O-imaging for myocardial perfusion and coronary angiography were performed. RESULTS: AdVEGF-B186 and AdVEGF-DΔNΔC induced angiogenesis and increased total microvascular area 1.8-fold (95% CI 0.2 to 3.5) and 2.8-fold (95% CI 1.4 to 4.3), respectively. At rest, perfusion was maintained at normal levels, but at stress, relative perfusion was increased 1.4-fold (95% CI 1.1 to 1.7) for AdVEGF-B186 and 1.3-fold (95% CI 1.0 to 1.7) for AdVEGF-DΔNΔC, without causing coronary steal effect in the control area. The therapy was well tolerated and did not lead to any significant changes in laboratory safety parameters. CONCLUSIONS: Both AdVEGF-B186 and AdVEGF-DΔNΔC gene transfers induced efficient angiogenesis in the myocardium resulting in an increased myocardial perfusion measured by PET. Importantly, local perfusion increase did not induce any coronary steal effect. As such, both treatments seem suitable new candidates for the induction of therapeutic angiogenesis for the treatment of refractory angina.

Effects of aging on angiogenesis.

Aging is a dominant risk factor for most forms of cardiovascular disease. Impaired angiogenesis and endothelial dysfunction likely contribute to the increased prevalence of both cardiovascular diseases and their adverse sequelae in the elderly. Angiogenesis is both an essential adaptive response to physiological stress and an endogenous repair mechanism after ischemic injury. In addition, induction of angiogenesis is a promising therapeutic approach for ischemic diseases. For these reasons, understanding the basis of age-related impairment of angiogenesis and endothelial function has important implications for understanding and managing cardiovascular disease. In this review, we discuss the molecular mechanisms that contribute to impaired angiogenesis in the elderly and potential therapeutic approaches to improving vascular function and angiogenesis in aging patients.

Growth factor therapy and autologous lymph node transfer in lymphedema.

BACKGROUND: Lymphedema after surgery, infection, or radiation therapy is a common and often incurable problem. Application of lymphangiogenic growth factors has been shown to induce lymphangiogenesis and to reduce tissue edema. The therapeutic effect of autologous lymph node transfer combined with adenoviral growth factor expression was evaluated in a newly established porcine model of limb lymphedema. METHODS AND RESULTS: The lymphatic vasculature was destroyed within a 3-cm radius around an inguinal lymph node. Lymph node grafts and adenovirally (Ad) delivered vascular endothelial growth factor (VEGF)-C (n=5) or VEGF-D (n=9) were used to reconstruct the lymphatic network in the inguinal area; AdLacZ (ß-galactosidase; n=5) served as a control. Both growth factors induced robust growth of new lymphatic vessels in the defect area, and postoperative lymphatic drainage was significantly improved in the VEGF-C/D-treated pigs compared with controls. The structure of the transferred lymph nodes was best preserved in the VEGF-C-treated pigs. Interestingly, VEGF-D transiently increased accumulation of seroma fluid in the operated inguinal region postoperatively, whereas VEGF-C did not have this side effect. CONCLUSIONS: These results show that growth factor gene therapy coupled with lymph node transfer can be used to repair damaged lymphatic networks in a large animal model and provide a basis for future clinical trials of the treatment of lymphedema.

VEGF-DdeltaNdeltaC mediated angiogenesis in skeletal muscles of diabetic WHHL rabbits.

BACKGROUND: Arterial occlusive disease is often associated with diabetes mellitus and hypercholesterolaemia which may reduce angiogenic potential of several growth factors. Accordingly, the usefulness of therapeutic angiogenesis in the presence of diabetes and hypercholesterolaemia has remained unclear. We evaluated angiogenic effects of the mature form of vascular endothelial growth factor-D (VEGF-D(deltaNdeltaC)) in skeletal muscles in the presence of severe diabetes and hypercholesterolaemia. METHODS: Intra muscular injections of adenoviruses encoding human VEGF-D(deltaNdeltaC) (AdVEGF-D(deltaNdeltaC)) were given in the hind limbs of a group of diabetic hypercholesterolaemic rabbits and adenoviruses encoding LacZ (AdLacZ) were used as a control. All animals were killed 6 days after the gene transfer. RESULTS: Capillary count, capillary area, capillary permeability and perfusion were significantly higher in the AdVEGF-D(deltaNdeltaC) transduced muscles compared with the AdLacZ controls. Expressions of endothelial nitric oxide synthase (eNOS) and VEGF receptor(R)-2 were also significantly increased in the VEGF-D(deltaNdeltaC) transduced muscles, along with an increased expression of angiopoietins (Angs) and neuropilin-2 (NP-2). Furthermore, VEGF-D(deltaNdeltaC) gene transfer to the skeletal muscles increased localized recruitment of cells with endothelial progenitor-like characteristics. CONCLUSIONS: VEGF-D(deltaNdeltaC) gene transfer can induce efficient angiogenesis in the presence of severe diabetes and hypercholesterolaemia by upregulating eNOS and VEGFR-2 expression. VEGF-D(deltaNdeltaC) appears to be a promising agent for inducing therapeutic angiogenesis even in cases with severe diabetes and hypercholesterolaemia.

Intravitreal adenoviral 15-lipoxygenase-1 gene transfer prevents vascular endothelial growth factor A-induced neovascularization in rabbit eyes.

Excessive angiogenesis mediated by vascular endothelial growth factor (VEGF) plays an important role in angioproliferative ocular diseases. We have previously developed a large animal model for these diseases by intravitreal adenoviral gene transfer of VEGF-A(165). 15-Lipoxygenase-1 (15-LO-1), an oxidizing enzyme producing reactive lipid hydroperoxides, has been shown to induce aberrant angiogenesis in cancer models of transgenic mice overexpressing human 15-LO-1. Our purpose was to study the effects of 15-LO-1 on VEGF-A(165)-induced angiogenesis in New Zealand White rabbit eyes, using intravitreal adenovirus-mediated gene transfers. AdCMV and Adh15-LO-1 alone served as controls. As determined by immunohistochemistry, VEGF-A(165) significantly increased the number and size of the capillaries in various compartments of the eyes. 15-LO-1 efficiently inhibited VEGF-A(165)-induced neovascularization and pathological changes by reducing VEGF-A(165) mRNA and protein expression, determined by RT-PCR, ELISA, and immunohistochemistry. 15-LO-1, which produces endogenous ligands for peroxisome proliferator-activated receptor-gamma (PPARgamma), also prevented VEGF-A(165)-induced expression of PPARgamma and VEGF receptor-2, as measured by quantitative RT-PCR. In conclusion, our findings show that 15-LO-1 prevents VEGF-A(165)-induced angiogenesis and consequent pathology in the eyes, suggesting that intravitreal 15-LO-1 gene transfer could be a potential new strategy for the treatment of neovascular complications in the eyes.

Vascular endothelial growth factor-B induces myocardium-specific angiogenesis and arteriogenesis via vascular endothelial growth factor receptor-1- and neuropilin receptor-1-dependent mechanisms.

BACKGROUND: New revascularization therapies are urgently needed for patients with severe coronary heart disease who lack conventional treatment options. METHODS AND RESULTS: We describe a new proangiogenic approach for these no-option patients using adenoviral (Ad) intramyocardial vascular endothelial growth factor (VEGF)-B186 gene transfer, which induces myocardium-specific angiogenesis and arteriogenesis in pigs and rabbits. After acute infarction, AdVEGF-B186 increased blood vessel area, perfusion, ejection fraction, and collateral artery formation and induced changes toward an ischemia-resistant myocardial phenotype. Soluble VEGF receptor-1 and soluble neuropilin receptor-1 reduced the effects of AdVEGF-B186, whereas neither soluble VEGF receptor-2 nor inhibition of nitric oxide production had this result. The effects of AdVEGF-B186 involved activation of neuropilin receptor-1, which is highly expressed in the myocardium, via recruitment of G-protein-alpha interacting protein, terminus C (GIPC) and upregulation of G-protein-alpha interacting protein. AdVEGF-B186 also induced an antiapoptotic gene expression profile in cardiomyocytes and had metabolic effects by inducing expression of fatty acid transport protein-4 and lipid and glycogen accumulation in the myocardium. CONCLUSIONS: VEGF-B186 displayed strikingly distinct effects compared with other VEGFs. These effects may be mediated at least in part via a G-protein signaling pathway. Tissue-specificity, high efficiency in ischemic myocardium, and induction of arteriogenesis and antiapoptotic and metabolic effects make AdVEGF-B186 a promising candidate for the treatment of myocardial ischemia.