Chunxia Cronin

Elevated Vascular Endothelial Growth Factor Receptor-2 Abundance Contributes to Increased Angiogenesis in Vascular Endothelial Growth Factor Receptor-1–Deficient Mice

Background —Vascular endothelial growth factor receptor-1 (VEGFR-1/Flt-1) is a potential therapeutic target for cardiovascular diseases, but its role in angiogenesis remains controversial. While germline Vegfr-1-/- embryos die of abnormal vascular development in association with excessive endothelial differentiation, mice lacking only the kinase domain are apparently healthy. Methods and Results —We carried out Cre- lox P mediated knockout to abrogate the expression of all known VEGFR-1 functional domains in neonatal and adult mice, and analyzed developmental, pathophysiological, and molecular consequences. VEGFR-1 deficiency promoted tip cell formation and endothelial cell (EC) proliferation, and facilitated angiogenesis of blood vessels which matured and perfused properly. Vascular permeability was normal at the basal level, but elevated in response to high doses of exogenous VEGF-A. In the post-infarct ischemic cardiomyopathy model, VEGFR-1 deficiency supported robust angiogenesis and protected against myocardial infarction. VEGFR-1 knockout led to abundant accumulation of VEGFR-2 at the protein level, increased VEGFR-2 tyrosine phosphorylation transiently, and enhanced serine phosphorylation of Akt and ERK. Interestingly, increased angiogenesis, tip cell formation, vascular permeability, VEGFR-2 accumulation, and Akt phosphorylation could be partially rescued or suppressed by one or more of the following manipulations, including injection of VEGFR-2 selective inhibitor SU1498, anti-VEGF-A, or introduction of Vegfr-2+/- heterozygosity into Vegfr-1 somatic knockout mice. Conclusions —Upregulation of VEGFR-2 abundance at the protein level contributes in part to increased angiogenesis in VEGFR-1 deficient mice.Background— Vascular endothelial growth factor receptor-1 (VEGFR-1/Flt-1) is a potential therapeutic target for cardiovascular diseases, but its role in angiogenesis remains controversial. Whereas germline Vegfr-1−/− embryos die of abnormal vascular development in association with excessive endothelial differentiation, mice lacking only the kinase domain appear healthy. Methods and Results— We performed Cre-loxP–mediated knockout to abrogate the expression of all known VEGFR-1 functional domains in neonatal and adult mice and analyzed developmental, pathophysiological, and molecular consequences. VEGFR-1 deficiency promoted tip cell formation and endothelial cell proliferation and facilitated angiogenesis of blood vessels that matured and perfused properly. Vascular permeability was normal at the basal level but elevated in response to high doses of exogenous VEGF-A. In the postinfarct ischemic cardiomyopathy model, VEGFR-1 deficiency supported robust angiogenesis and protected against myocardial infarction. VEGFR-1 knockout led to abundant accumulation of VEGFR-2 at the protein level, increased VEGFR-2 tyrosine phosphorylation transiently, and enhanced serine phosphorylation of Akt and ERK. Interestingly, increased angiogenesis, tip cell formation, vascular permeability, VEGFR-2 accumulation, and Akt phosphorylation could be partially rescued or suppressed by one or more of the following manipulations, including injection of the VEGFR-2 selective inhibitor SU1498, anti-VEGF-A, or introduction of Vegfr-2+/− heterozygosity into Vegfr-1 somatic knockout mice. Conclusions— Upregulation of VEGFR-2 abundance at the protein level contributes in part to increased angiogenesis in VEGFR-1–deficient mice.

Role of P2X purinergic receptors in the rescue of ischemic heart failure

Evidence is accumulating to support the presence of P2X purinergic receptors in the heart. However, the biological role of this receptor remains to be defined. The objectives here were to determine the role of cardiac P2X receptors in modulating the progression of post-myocardial infarction ischemic heart failure and to investigate the underlying mechanism. The P2X4 receptor (P2X4R) is an important subunit of native cardiac P2X receptors, and the cardiac-specific transgenic overexpression of P2X4R (Tg) was developed as a model. Left anterior descending artery ligation resulted in similar infarct size between Tg and wild-type (WT) mice (P > 0.1). However, Tg mice showed an enhanced cardiac contractile performance at 7 days, 1 mo, and 2 mo after infarction and an increased survival at 1 and 2 mo after infarction (P < 0.01). The enhanced intact heart function was manifested by a greater global left ventricular developed pressure and rate of contraction of left ventricular pressure in vitro and by a significantly increased fractional shortening and systolic thickening in the noninfarcted region in vivo (P < 0.05). The salutary effects on the ischemic heart failure phenotype were seen in both sexes and were not the result of any difference in infarct size in Tg versus WT hearts. An enhanced contractile function of the noninfarcted area in the Tg heart was likely an important rescuing mechanism. The cardiac P2X receptor is a novel target to treat post-myocardial infarction ischemic heart failure.

CD13 is essential for inflammatory trafficking and infarct healing following permanent coronary artery occlusion in mice

Abstract Aims To determine the role of CD13 as an adhesion molecule in trafficking of inflammatory cells to the site of injury in vivo and its function in wound healing following myocardial infarction induced by permanent coronary artery occlusion. Methods and results Seven days post-permanent ligation, hearts from CD13 knockout (CD13KO) mice showed significant reductions in cardiac function, suggesting impaired healing in the absence of CD13. Mechanistically, CD13KO infarcts showed an increase in small, endothelial-lined luminal structures, but no increase in perfusion, arguing against an angiogenic defect in the absence of CD13. Cardiac myocytes of CD13KO mice showed normal basal contractile function, eliminating myocyte dysfunction as a mechanism of adverse remodelling. Conversely, immunohistochemical and flow cytometric analysis of CD13KO infarcts demonstrated a dramatic 65% reduction in infiltrating haematopoietic cells, including monocytes, macrophages, dendritic, and T cells, suggesting a critical role for CD13 adhesion in inflammatory trafficking. Accordingly, CD13KO infarcts also contained fewer myofibroblasts, consistent with attenuation of fibroblast differentiation resulting from the reduced inflammation, leading to adverse remodelling. Conclusion In the ischaemic heart, while compensatory mechanisms apparently relieve potential angiogenic defects, CD13 is essential for proper trafficking of the inflammatory cells necessary to prime and sustain the reparative response, thus promoting optimal post-infarction healing.

Elevated VEGF Receptor-2 Abundance Contributes to Increased Angiogenesis in VEGF Receptor-1 Deficient Mice

Background —Vascular endothelial growth factor receptor-1 (VEGFR-1/Flt-1) is a potential therapeutic target for cardiovascular diseases, but its role in angiogenesis remains controversial. While germline Vegfr-1-/- embryos die of abnormal vascular development in association with excessive endothelial differentiation, mice lacking only the kinase domain are apparently healthy. Methods and Results —We carried out Cre- lox P mediated knockout to abrogate the expression of all known VEGFR-1 functional domains in neonatal and adult mice, and analyzed developmental, pathophysiological, and molecular consequences. VEGFR-1 deficiency promoted tip cell formation and endothelial cell (EC) proliferation, and facilitated angiogenesis of blood vessels which matured and perfused properly. Vascular permeability was normal at the basal level, but elevated in response to high doses of exogenous VEGF-A. In the post-infarct ischemic cardiomyopathy model, VEGFR-1 deficiency supported robust angiogenesis and protected against myocardial infarction. VEGFR-1 knockout led to abundant accumulation of VEGFR-2 at the protein level, increased VEGFR-2 tyrosine phosphorylation transiently, and enhanced serine phosphorylation of Akt and ERK. Interestingly, increased angiogenesis, tip cell formation, vascular permeability, VEGFR-2 accumulation, and Akt phosphorylation could be partially rescued or suppressed by one or more of the following manipulations, including injection of VEGFR-2 selective inhibitor SU1498, anti-VEGF-A, or introduction of Vegfr-2+/- heterozygosity into Vegfr-1 somatic knockout mice. Conclusions —Upregulation of VEGFR-2 abundance at the protein level contributes in part to increased angiogenesis in VEGFR-1 deficient mice.Background— Vascular endothelial growth factor receptor-1 (VEGFR-1/Flt-1) is a potential therapeutic target for cardiovascular diseases, but its role in angiogenesis remains controversial. Whereas germline Vegfr-1−/− embryos die of abnormal vascular development in association with excessive endothelial differentiation, mice lacking only the kinase domain appear healthy. Methods and Results— We performed Cre-loxP–mediated knockout to abrogate the expression of all known VEGFR-1 functional domains in neonatal and adult mice and analyzed developmental, pathophysiological, and molecular consequences. VEGFR-1 deficiency promoted tip cell formation and endothelial cell proliferation and facilitated angiogenesis of blood vessels that matured and perfused properly. Vascular permeability was normal at the basal level but elevated in response to high doses of exogenous VEGF-A. In the postinfarct ischemic cardiomyopathy model, VEGFR-1 deficiency supported robust angiogenesis and protected against myocardial infarction. VEGFR-1 knockout led to abundant accumulation of VEGFR-2 at the protein level, increased VEGFR-2 tyrosine phosphorylation transiently, and enhanced serine phosphorylation of Akt and ERK. Interestingly, increased angiogenesis, tip cell formation, vascular permeability, VEGFR-2 accumulation, and Akt phosphorylation could be partially rescued or suppressed by one or more of the following manipulations, including injection of the VEGFR-2 selective inhibitor SU1498, anti-VEGF-A, or introduction of Vegfr-2+/− heterozygosity into Vegfr-1 somatic knockout mice. Conclusions— Upregulation of VEGFR-2 abundance at the protein level contributes in part to increased angiogenesis in VEGFR-1–deficient mice.

Two alanines juxtaposed to aggrecan's G1 domain alter its intracellular localization

Nascent proteins translated and processed in the endoplasmic reticulum (ER) sometimes contain intrinsic signals for ER retention or ER retrieval. These signals are usually a few amino acids in length, and if alanine modifications are made within these sequences, normal transit patterns of the nascent protein frequently change. The purpose of this study was to determine whether two alanines juxtaposed to the first globular domain of aggrecans core protein affect its transit in Chinese hamster ovary (CHO) cells. Results show that two alanines juxtaposed to the first globular domain (G1AA) minimized secretion of the protein. However, transgenic proteins with juxtaposed glutamate–phenylalanine (G1EF) or no additional amino acids (G1) were still secreted. GFP‐tagged G1AA localized in the lumen of the ER but not in the Golgi. In contrast, a portion of GFP‐tagged G1EF and G1 did appear in the Golgi compartment. More importantly, unique and striking accumulations of G1EF and G1 transgenic proteins were seen in large dilated regions of the ER cisternae, reminiscent of accumulations seen in α1‐antitrypsin deficiency disease. G1AA transgenic proteins did not form these vesicles but were diffusely distributed throughout the ER lumen. These results indicate that just two juxtaposed alanines can profoundly affect a large globular proteins intracellular localization.

Deletion of the P2X4 receptor is neuroprotective acutely, but induces a depressive phenotype during recovery from ischemic stroke

INTRODUCTION Acute ischemic injury leads to severe neuronal loss. One of the key mechanisms responsible for this effect is inflammation, which is characterized by the activation of myeloid cells, including resident microglia and infiltrating monocytes/macrophages. P2X4 receptors (P2X4Rs) present on these immune cells modulate the inflammatory response. For example, excessive release of adenosine triphosphate during acute ischemic stroke triggers stimulation of P2X4Rs, leading to myeloid cell activation and proliferation and further exacerbating post-ischemic inflammation. In contrast, during recovery P2X4Rs activation on microglia leads to the release of brain-derived neurotrophic factor (BDNF), which alleviate depression, maintain synaptic plasticity and hasten post-stroke behavioral recovery. Therefore, we hypothesized that deletion of the P2X4R specifically from myeloid cells would have differential effects on acute versus chronic recovery following stroke. METHODS We subjected global or myeloid-specific (MS) P2X4R knock-out (KO) mice and wild-type littermates of both sexes to right middle cerebral artery occlusion (60min). We performed histological, behavioral (sensorimotor and depressive), and biochemical (quantitative PCR and flow cytometry) analyses to determine the acute (three days after occlusion) and chronic (30days after occlusion) effects of receptor deletion. RESULTS Global P2X4R deletion led to reduced infarct size in both sexes. In MS P2X4R KO mice, only females showed reduced infarct size, an effect that did not change with ovariectomy. MS P2X4R KO mice of both sexes showed swift recovery from sensorimotor deficits during acute recovery but exhibited a more pronounced post-stroke depressive behavior phenotype that was independent of infarct size. Quantitative PCR analysis of whole cell lysate as well as flow-sorted myeloid cells from the perilesional cortex showed increased cellular interleukin 1 beta (IL-1β), interleukin 6 (IL-6), and tumor necrosis factor alpha (TNF-α) mRNA levels but reduced plasma levels of these cytokines in MS P2X4R KO mice after stroke. The expression levels of BDNF and other depression-associated genes were reduced in MS P2X4R KO mice after stroke. CONCLUSIONS P2X4R deletion protects against stroke acutely but predisposes to depression-like behavior chronically after stroke. Thus, a time-sensitive approach should be considered when targeting P2X4Rs after stroke.

Elevated Vascular Endothelial Growth Factor Receptor-2 Abundance Contributes to Increased Angiogenesis in Vascular Endothelial Growth Factor Receptor-1–Deficient MiceCLINICAL PERSPECTIVE

Background —Vascular endothelial growth factor receptor-1 (VEGFR-1/Flt-1) is a potential therapeutic target for cardiovascular diseases, but its role in angiogenesis remains controversial. While germline Vegfr-1-/- embryos die of abnormal vascular development in association with excessive endothelial differentiation, mice lacking only the kinase domain are apparently healthy. Methods and Results —We carried out Cre- lox P mediated knockout to abrogate the expression of all known VEGFR-1 functional domains in neonatal and adult mice, and analyzed developmental, pathophysiological, and molecular consequences. VEGFR-1 deficiency promoted tip cell formation and endothelial cell (EC) proliferation, and facilitated angiogenesis of blood vessels which matured and perfused properly. Vascular permeability was normal at the basal level, but elevated in response to high doses of exogenous VEGF-A. In the post-infarct ischemic cardiomyopathy model, VEGFR-1 deficiency supported robust angiogenesis and protected against myocardial infarction. VEGFR-1 knockout led to abundant accumulation of VEGFR-2 at the protein level, increased VEGFR-2 tyrosine phosphorylation transiently, and enhanced serine phosphorylation of Akt and ERK. Interestingly, increased angiogenesis, tip cell formation, vascular permeability, VEGFR-2 accumulation, and Akt phosphorylation could be partially rescued or suppressed by one or more of the following manipulations, including injection of VEGFR-2 selective inhibitor SU1498, anti-VEGF-A, or introduction of Vegfr-2+/- heterozygosity into Vegfr-1 somatic knockout mice. Conclusions —Upregulation of VEGFR-2 abundance at the protein level contributes in part to increased angiogenesis in VEGFR-1 deficient mice.Background— Vascular endothelial growth factor receptor-1 (VEGFR-1/Flt-1) is a potential therapeutic target for cardiovascular diseases, but its role in angiogenesis remains controversial. Whereas germline Vegfr-1−/− embryos die of abnormal vascular development in association with excessive endothelial differentiation, mice lacking only the kinase domain appear healthy. Methods and Results— We performed Cre-loxP–mediated knockout to abrogate the expression of all known VEGFR-1 functional domains in neonatal and adult mice and analyzed developmental, pathophysiological, and molecular consequences. VEGFR-1 deficiency promoted tip cell formation and endothelial cell proliferation and facilitated angiogenesis of blood vessels that matured and perfused properly. Vascular permeability was normal at the basal level but elevated in response to high doses of exogenous VEGF-A. In the postinfarct ischemic cardiomyopathy model, VEGFR-1 deficiency supported robust angiogenesis and protected against myocardial infarction. VEGFR-1 knockout led to abundant accumulation of VEGFR-2 at the protein level, increased VEGFR-2 tyrosine phosphorylation transiently, and enhanced serine phosphorylation of Akt and ERK. Interestingly, increased angiogenesis, tip cell formation, vascular permeability, VEGFR-2 accumulation, and Akt phosphorylation could be partially rescued or suppressed by one or more of the following manipulations, including injection of the VEGFR-2 selective inhibitor SU1498, anti-VEGF-A, or introduction of Vegfr-2+/− heterozygosity into Vegfr-1 somatic knockout mice. Conclusions— Upregulation of VEGFR-2 abundance at the protein level contributes in part to increased angiogenesis in VEGFR-1–deficient mice.