Neural JNK3 regulates blood flow recovery after hindlimb ischemia in mice via an Egr1/Creb1 axis.

Diseases related to impaired blood flow such as peripheral artery disease (PAD) impact nearly 10 million people in the United States alone, yet patients with clinical manifestations of PAD (e.g., claudication and limb ischemia) have limited treatment options. In ischemic tissues, stress kinases such as c-Jun N-terminal kinases (JNKs), are activated. Here, we show that inhibition of the JNK3 (Mapk10) in the neural compartment strikingly potentiates blood flow recovery from mouse hindlimb ischemia. JNK3 deficiency leads to upregulation of growth factors such as Vegfa, Pdgfb, Pgf, Hbegf and Tgfb3 in ischemic muscle by activation of the transcription factors Egr1/Creb1. JNK3 acts through Forkhead box O3 (Foxo3a) to suppress the activity of Egr1/Creb1 transcription regulators in vitro. In JNK3-deficient cells, Foxo3a is suppressed which leads to Egr1/Creb1 activation and upregulation of downstream growth factors. Collectively, these data suggest that the JNK3-Foxo3a-Egr1/Creb1 axis coordinates the vascular remodeling response in peripheral ischemia.

Suppression of ischemia in arterial occlusive disease by JNK-promoted native collateral artery development.

Arterial occlusive diseases are major causes of morbidity and mortality. Blood flow to the affected tissue must be restored quickly if viability and function are to be preserved. We report that disruption of the mixed-lineage protein kinase (MLK) - cJun NH2-terminal kinase (JNK) signaling pathway in endothelial cells causes severe blockade of blood flow and failure to recover in the murine femoral artery ligation model of hindlimb ischemia. We show that the MLK-JNK pathway is required for the formation of native collateral arteries that can restore circulation following arterial occlusion. Disruption of the MLK-JNK pathway causes decreased Dll4/Notch signaling, excessive sprouting angiogenesis, and defects in developmental vascular morphogenesis. Our analysis demonstrates that the MLK-JNK signaling pathway is a key regulatory mechanism that protects against ischemia in arterial occlusive disease.

A non membrane-targeted human soluble CD59 attenuates choroidal neovascularization in a model of age related macular degeneration.

Age related macular degeneration (AMD) is the most common cause of blindness amongst the elderly. Approximately 10% of AMD patients suffer from an advanced form of AMD characterized by choroidal neovascularization (CNV). Recent evidence implicates a significant role for complement in the pathogenesis of AMD. Activation of complement terminates in the incorporation of the membrane attack complex (MAC) in biological membranes and subsequent cell lysis. Elevated levels of MAC have been documented on choroidal blood vessels and retinal pigment epithelium (RPE) of AMD patients. CD59 is a naturally occurring membrane bound inhibitor of MAC formation. Previously we have shown that membrane bound human CD59 delivered to the RPE cells of mice via an adenovirus vector can protect those cells from human complement mediated lysis ex vivo. However, application of those observations to choroidal blood vessels are limited because protection from MAC- mediated lysis was restricted only to the cells originally transduced by the vector. Here we demonstrate that subretinal delivery of an adenovirus vector expressing a transgene for a soluble non-membrane binding form of human CD59 can attenuate the formation of laser-induced choroidal neovascularization and murine MAC formation in mice even when the region of vector delivery is distal to the site of laser induced CNV. Furthermore, this same recombinant transgene delivered to the intravitreal space of mice by an adeno-associated virus vector (AAV) can also attenuate laser-induced CNV. To our knowledge, this is the first demonstration of a non-membrane targeting CD59 having biological potency in any animal model of disease in vivo. We propose that the above approaches warrant further exploration as potential approaches for alleviating complement mediated damage to ocular tissues in AMD.

Expression of complement component 3 (C3) from an adenovirus leads to pathology in the murine retina.

PURPOSE: Activation of complement has been implicated as one of the major causes of age-related macular degeneration (AMD). Evidence is accumulating for a role of complement in other retinal diseases, such as diabetic retinopathy and proliferative vitreoretinopathy. Because of the paucity of animal models that directly investigate the role of complement in retinal pathology, the authors sought to develop a model of increased complement expression and activation, specifically in the murine retina. METHODS: The authors constructed a recombinant adenovirus-expressing murine complement component 3 (C3, AdcmvC3). Adult mice were injected in the subretinal space with either AdcmvC3 or a control virus, AdcmvGFP. After 1 to 2 weeks of exogenous C3 expression, mice were analyzed by scotopic electroretinography and fluorescein angiography. Eyes were harvested for histologic, immunohistochemical, and quantitative RT-PCR analyses. RESULTS: Mice injected with C3-expressing adenovirus exhibited significantly increased vascular permeability, endothelial cell proliferation and migration, RPE atrophy, loss of photoreceptor outer segments, reactive gliosis, retinal detachment, and reduced retinal function relative to those injected with a control adenovirus. Deposition of the membrane attack complex was observed on endothelial cells and photoreceptor outer segments. CONCLUSIONS: Adenovirus-mediated delivery of C3 to murine RPE induces significant functional and anatomic changes that reproduce many of the features of AMD as well as those of other retinal diseases. This novel model may be useful in assessing the role of complement in retinal pathology and in developing anti-complement therapies for retinal diseases associated with complement activation.

Evaluation of adenovirus-delivered human CD59 as a potential therapy for AMD in a model of human membrane attack complex formation on murine RPE.

PURPOSE: Complement-mediated damage to the retinal pigment epithelium (RPE), Bruch membrane, and choroid has been associated with pathogenesis in age-related macular degeneration (AMD). The terminal step of complement activation involves lysis of cells by the insertion of the membrane attack complex (MAC) in the plasma membrane. The hypothesis that local overexpression of human CD59 (hCD59) delivered by an adenovirus (Ad) vector to primary murine RPE cells in vitro, RPE in vivo, or cornea ex vivo protects those cells from human MAC deposition and lysis was tested. METHODS: A humanized model of MAC deposition on murine cells and murine ocular tissues including RPE and cornea was developed to permit testing of human complement regulators in mice. A recombinant adenovirus-expressing hCD59 was generated, and this virus was injected into the subretinal space of adult mice. Subsequently, eyecups from these mice were exposed to human serum, and the levels of MAC deposition on the RPE were quantified. hCD59 was also expressed on murine cornea ex vivo and in murine hepatocytes, and primary RPE cells in vitro and levels of human MAC deposition and cell lysis were measured. RESULTS: Adenovirus-mediated delivery of hCD59 to the RPE, cornea, or cells in culture protects those cells from human MAC deposition and MAC-mediated damage and vesiculation. CONCLUSIONS: The humanized model of MAC deposition on murine ocular tissues allows testing of human complement regulators that may have potential in the treatment of AMD or other diseases associated with complement activation.