Jun Kinases in Hematopoiesis, and Vascular Development and Function: A Dissertation

Arterial occlusive diseases are major causes of morbidity and mortality in industrialized countries and represent a huge economic burden. The extent of the native collateral circulation is an important determinant of blood perfusion restoration and therefore the severity of tissue damage and functional impairment that ensues following arterial occlusion. Understanding the mechanisms responsible for collateral artery development may provide avenues for therapeutic intervention. Here, we identify a critical requirement for mixed lineage kinase (MLK) – cJun-NH2-terminal kinase (JNK) signaling in vascular morphogenesis and native collateral artery development. We demonstrate that Mlk2-/-Mlk3-/- mice or mice with compound JNK-deficiency in the vascular endothelium display abnormal collateral arteries, which are unable to restore blood perfusion following arterial occlusion, leading to severe tissue necrosis in animal models of femoral and coronary artery occlusion. Employing constitutive and inducible conditional deletion strategies, we demonstrate that endothelial JNK acts during the embryonic development of collateral arteries to ensure proper patterning and maturation, but is dispensable for angiogenic and arteriogenic responses in adult mice. During developmental vascular morphogenesis, MLK – JNK signaling is required for suppression of excessive sprouting angiogenesis likely via JNK-dependent regulation of Dll4 expression and Notch signaling. This function of JNK may underlie its critical requirement for native collateral artery formation. Thus, this study introduces MLK – JNK signaling as a major regulator of vascular development. In contrast, we find that JNK in hematopoietic cells, which are thought to share a common mesodermally-derived precursor with endothelial cells, is cellautonomously dispensable for normal hematopoietic development and hematopoietic stem cell self-renewal, illustrating the highly context dependent function of JNK

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

AdCAGsCD59-conditioned media confers significant protection from human serum-mediated cell lysis.

<p>(A) A representative FACS histogram for hepa-1c1c7 cells treated with either NHS or HI-NHS in media conditioned with either AdCAGGFP or AdCAGsCD59. (B) While no significant difference in cell lysis was observed in hepa-1c1c7 cells treated with HI-NHS in media conditioned with either AdCAGGFP or AdCAGsCD59, a significant (34.08±6.40%, **p<0.01) reduction was observed in cell lysis in hepa-1c1c7 cells treated with NHS in media conditioned with AdCAGsCD59 relative to those cells treated with NHS in media conditioned with the control virus, AdCAGGFP. HI-NHS, heat-inactivated normal human serum; NHS, normal human serum. n = 4.</p

AdCAGpA and AdCAGsCD59 coinjected with AdCAGGFP result in equivalent levels of transduction and at sites distal to the sites of laser burn.

<p>Representative micrographs showing RPE/choroid flatmounts transduced with AdCAGGFP:AdCAGpA (1∶10) and AdCAGGFP:AdCAGsCD59 (1∶10). The region of transduction is demarcated and each of the three laser spots labeled 1–3. No significant difference in area of transduction was observed between AdCAGpA-injected and AdCAGsCD59-injected eyecups. n = 10 eyes (AdCAGsCD59), n = 8 eyes (AdCAGpA). ns, not significant. Scale bar = 200 µm.</p

sCD59 is expressed and efficiently secreted from adenovirus <i>in vitro.</i>

<p>(A) Expression cassettes for both sCD59 and GFP or without a transgene were cloned into the deleted E1 region of an E1/E3-deleted adenovirus. (B) ARPE-19 cells infected with AdCAGsCD59 express sCD59 and exhibit robust secretion into the media. CAG, chicken b-actin promoter; pA, polyA; LITR/RITR, left/right inverted terminal repeat; ¥, packaging signal; ΔE1/E3, deleted early regions 1/3; E2/E4, early regions 2/4.</p

Delivery of adenovirus to mouse subretinal space does not affect size of laser-induced CNV.

<p>Representative micrographs of FITC-GSL I stained laser-induced CNV spots from each of AdCAGpA-injected and uninjected eyecups. No significant difference was observed in the size of spots induced by laser in AdCAGpA-injected relative to uninjected eyes (p>0.5). n = 12 spots/5 eyes (uninjected), n = 26 spots/9 eyes (AdCAGpA); ns, not significant. Scale bar = 50 µm.</p

AdCAGsCD59 delivered to murine RPE results in a significant reduction in laser-induced CNV.

<p>Representative micrographs showing FITC-GSL I stained laser-induced CNV spots from eyes injected with either AdCAGpA or AdCAGsCD59. AdCAGsCD59 transduction of murine RPE results in a 61.7±19.9% reduction in size of CNV spot relative to eyes injected with AdCAGpA (**p<0.01). n = 33 spots/12 eyes (AdCAGsCD59), n = 26 spots/9 eyes (AdCAGpA). Scale bar = 50 µm.</p

Intravitreal delivery of an AAV expressing sCD59 results in a significant protection against laser-induced CNV.

<p>(A) The expression cassette containing sCD59 under the control of the chicken β-actin promoter was cloned into an AAV serotype 2 vector. A control virus expressing GFP was also generated. (B) Western blot analysis confirms secretion of sCD59 by human embryonic retinoblasts infected with AAVCAGsCD59. (C) Representative micrographs of laser-induced CNV spots observed in AAVCAGGFP-injected and AAVCAGsCD59-injected eyecups. (D) A significant 62.6±16.9% (***p<0.001) reduction in size of CNV was observed in AAVCAGsCD59-injected eyecups relative to those injected with AAVCAGGFP. n = 42 spots/11 eyes (AAVCAGGFP), n = 53 spots/14 eyes (AAVCAGsCD59). Scale bar = 50 µm.</p

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