Dan Chalothorn

Vascular Endothelial Growth Factor-A Specifies Formation of Native Collaterals and Regulates Collateral Growth in Ischemia

The density of native (preexisting) collaterals and their capacity to enlarge into large conduit arteries in ischemia (arteriogenesis) are major determinants of the severity of tissue injury in occlusive disease. Mechanisms directing arteriogenesis remain unclear. Moreover, nothing is known about how native collaterals form in healthy tissue. Evidence suggests vascular endothelial growth factor (VEGF), which is important in embryonic vascular patterning and ischemic angiogenesis, may contribute to native collateral formation and arteriogenesis. Therefore, we examined mice heterozygous for VEGF receptor-1 (VEGFR-1+/−), VEGF receptor-2 (VEGFR-2+/−), and overexpressing (VEGFhi/+) and underexpressing VEGF-A (VEGFlo/+). Recovery from hindlimb ischemia was followed for 21 days after femoral artery ligation. All statements below are P<0.05. Compared to wild-type mice, VEGFR-2+/− showed similar: ischemic scores, recovery of hindlimb perfusion, pericollateral leukocytes, collateral enlargement, and angiogenesis. In contrast, VEGFR-1+/− showed impaired: perfusion recovery, pericollateral leukocytes, collateral enlargement, worse ischemic scores, and comparable angiogenesis. Compared to wild-type mice, VEGFlo/+ had 2-fold lower perfusion immediately after ligation (suggesting fewer native collaterals which was confirmed by angiography) and blunted recovery of perfusion. VEGFhi/+ mice had 3-fold greater perfusion immediately after ligation, more native collaterals, and improved recovery of perfusion. These differences were confirmed in the cerebral pial cortical circulation where, compared to VEGFhi/+ mice, VEGFlo/+ formed fewer collaterals during the perinatal period when adult density was established, and had 2-fold larger infarctions after middle cerebral artery ligation. Our findings indicate VEGF and VEGFR-1 are determinants of arteriogenesis. Moreover, we describe the first signaling molecule, VEGF-A, that specifies formation of native collaterals in healthy tissues.

Transactivation of Epidermal Growth Factor Receptor Mediates Catecholamine-Induced Growth of Vascular Smooth Muscle

Stimulation of &agr;1-adrenoceptors induces proliferation of vascular smooth muscle cells (SMCs) and contributes to arterial remodeling. Although activation of NAD(P)H oxidase and generation of reactive oxygen species (ROS) are required, little is known about this pathway. In this study, we examined the hypothesis that epidermal growth factor receptor (EGFR) transactivation and extracellular regulated kinases (ERK) are involved in &agr;1-adrenoceptor–mediated SMC growth. Phenylephrine increased protein synthesis in association with a rapid (≤5 minutes) and sustained (≥60 minutes) doubling of phosphorylation of EGFR and ERK1/2, but not p38 or JNK in the media of rat aorta maintained in organ culture. Antagonists of EGFR phosphotyrosine activity (AG-1478) and ERK phosphorylation (PD-98059, U-0126) abolished phenylephrine-induced protein synthesis, whereas antagonists of p38 or JNK phosphorylation had no specific effect. A competitive antagonist (P22) for heparin binding EGF-like growth factor (HB-EGF) blocked phenylephrine-induced protein synthesis, as did downregulation of pro-HB-EGF (CRM197). Phenylephrine-induced protein synthesis was inhibited by neutralizing antibody to HB-EGF and absent in HB-EGF−/− SMCs. Inhibitors of metalloproteinases (BiPS, KB-R7785) also blocked adrenergic growth. The neutralizing antibody against HB-EGF had no effect on the two-fold increase in ROS generation induced by phenylephrine (DCF fluorescence), suggesting that stimulation of NAD(P)H oxidase by &agr;1-adrenoceptor occupation precedes HB-EGF release. Cell culture studies confirmed and extended these findings. These data suggest that &agr;1-adrenoceptor–mediated SMC growth requires ROS-dependent shedding of HB-EGF, transactivation of EGFR, and activation of the MEK1/2-dependent MAP kinase pathway. This trophic pathway may link sympathetic activity to arterial wall growth in adaptive remodeling and hypertrophic disease.

Strain-dependent variation in collateral circulatory function in mouse hindlimb

The extent (density and diameter) of the native (preexisting) collateral circulation in healthy tissues and the capacity of collaterals to enlarge/remodel in obstructive arterial disease are important determinants of ischemic injury. Evidence suggests that these parameters vary widely from yet-to-be-identified genetic and environmental factors. Recently, a locus on chromosome 7 was linked to less recovery of perfusion after femoral artery ligation in BALB/c and A/J versus C57BL/6 mouse strains. Moreover, evidence suggested that BALB/c and A/J share an allele(s) at this locus that is different from C57BL/6 mice. Here we tested the hypothesis that differences in collateral extent and/or remodeling underlie these findings. Compared with C57BL/6, BALB/c and A/J strains have fewer native collaterals in hindlimb (also confirmed in brain)-associated with greater reduction in perfusion immediately after femoral ligation, slower recovery of perfusion, greater hindlimb use impairment, and worse ischemia. However, A/J also differed from BALB/c in a number of these parameters, including having more robust collateral remodeling. Analysis of A/J --> C57BL/6 chromosome substitution strains confirmed that a difference in an allele(s) on chromosome 7 conferred most, but not all, of the magnitude of the differences in collateral function. Additional studies of C57BL/6 x BALB/c F1 mice demonstrated that alleles of the C57BL/6 strain exert dominance for collateral traits. Finally, negative results were obtained from studies examining a previously identified candidate gene potentially responsible for these differences-Bcl2-associated athanogene-3. These findings emphasize the major contribution of genetic background to variation in the collateral circulation and its capacity to lessen ischemia in obstructive disease.

Chloride Intracellular Channel-4 Is a Determinant of Native Collateral Formation in Skeletal Muscle and Brain

The capacity of the collateral circulation to lessen injury in occlusive vascular disease depends on the density and caliber of native (preexisting) collaterals, as well as their ability to outwardly remodel in ischemia. Native collateral conductance varies widely among healthy individuals, yet little is known about what specifies collateral formation. Chloride intracellular channel (CLIC)4 protein is required for endothelial cell hollowing, a process necessary for vessel formation during embryogenesis and ischemia. Whether CLIC4 has other physiological roles in vascular biology is uncertain. We studied collateral formation and remodeling in mice deficient in CLIC1 and CLIC4. Vascular responses to femoral artery ligation were similar in Clic1−/− and wild-type mice. In contrast, immediately after ligation perfusion dropped more in Clic4−/− than wild-type mice, suggesting fewer preexisting collaterals, a finding confirmed by angiography, greater ischemia, and worse recovery of perfusion; however, collateral remodeling was unaffected. Likewise, native cerebral collateral density in Clic4−/− (but not Clic1−/−) mice was reduced, resulting in severe infarctions. This was associated with impaired perinatal formation and stabilization of nascent collaterals. Clic4 hemizygous mice had intermediate deficits in the above parameters, suggesting a gene–dose effect. Ischemia augmented CLIC1 and CLIC4 expression similarly in wild-type mice. However, CLIC1 increased 3-fold more in Clic4−/− mice, suggesting compensation. Despite greater ischemia in Clic4−/− mice, hypoxia-inducible factor-1α, vascular endothelial growth factor (VEGF) and angiopoietin-2 increased less compared to wild-type, suggesting CLIC4 exerts influences upstream of hypoxia-inducible factor-1α–VEGF signaling. Hence, CLIC4 represents the second gene that, along with VEGF shown by us previously, specifies native collateral formation.

Heparin-Binding Epidermal Growth Factor–Like Growth Factor, Collateral Vessel Development, and Angiogenesis in Skeletal Muscle Ischemia

Objective—Heparin-binding epidermal growth factor–like growth factor (HB-EGF) is a potent mitogen for smooth muscle cells and has been implicated in atherosclerosis, tissue regeneration after ischemia, vascular development, and tumor angiogenesis. We examined the hypothesis that HB-EGF participates in angiogenesis and collateral growth in ischemia. Methods and Results—During 3 weeks after femoral artery ligation, no attenuation occurred in recovery of hindlimb perfusion or distal saphenous artery flow in HB-EGF–null (HB-EGF−/−) versus wild-type mice. Lumen diameters of remodeled collaterals in gracilis muscle were similar by morphometry (87±8 versus 94±6 &mgr;m) and angiography, although medial thickening was reduced. Gastrocnemius muscle underwent comparable angiogenesis (41% and 33% increase in capillary-to-muscle fiber ratio). Renal renin mRNA, arterial pressure, and heart rate during anesthesia or conscious unrestrained conditions were similar between groups. These latter findings validate comparisons of perfusion data and also suggest that differences in arterial pressure and/or renin–angiotensin activity are not masking an otherwise inhibitory effect of HB-EGF absence. Four days after ligation, EGF receptor phosphorylation increased in muscle by 104% in wild-type but by only 30% in HB-EGF−/− mice. This argues against compensation by other EGF receptor ligands. Conclusion—Our results suggest that HB-EGF is not required for arteriogenesis or angiogenesis in hindlimb ischemia.

CIB1 Regulates Endothelial Cells and Ischemia-Induced Pathological and Adaptive Angiogenesis

Pathological angiogenesis contributes to various ocular, malignant, and inflammatory disorders, emphasizing the need to understand this process on a molecular level. CIB1 (calcium- and integrin-binding protein), a 22-kDa EF-hand–containing protein, modulates the activity of p21-activated kinase 1 in fibroblasts. Because p21-activated kinase 1 also contributes to endothelial cell function, we hypothesized that CIB1 may have a role in angiogenesis. We found that endothelial cells depleted of CIB1 by either short hairpin RNA or homologous recombination have reduced migration, proliferation, and tubule formation. Moreover, loss of CIB1 in these cells decreases p21-activated kinase 1 activation, downstream extracellular signal-regulated kinase 1/2 activation, and matrix metalloproteinase 2 expression, all of which are known to contribute to angiogenesis. Consistent with these findings, tissues derived from CIB1-deficient (CIB1−/−) mice have reduced growth factor–induced microvessel sprouting in ex vivo organ cultures and in vivo Matrigel plugs. Furthermore, in response to ischemia, CIB1−/− mice demonstrate decreased pathological retinal and adaptive hindlimb angiogenesis. Ischemic CIB1−/− hindlimbs also demonstrate increased tissue damage and significantly reduced p21-activated kinase 1 activation. These data therefore reveal a critical role for CIB1 in ischemia-induced pathological and adaptive angiogenesis.

Tumor growth and angiogenesis is impaired in CIB1 knockout mice

BackgroundPathological angiogenesis contributes to various ocular, malignant, and inflammatory disorders, emphasizing the need to understand this process more precisely on a molecular level. Previously we found that CIB1, a 22 kDa regulatory protein, plays a critical role in endothelial cell function, angiogenic growth factor-mediated cellular functions, PAK1 activation, MMP-2 expression, and in vivo ischemia-induced angiogenesis. Since pathological angiogenesis is highly dependent on many of these same processes, we hypothesized that CIB1 may also regulate tumor-induced angiogenesis.MethodsTo test this hypothesis, we allografted either murine B16 melanoma or Lewis lung carcinoma cells into WT and CIB1-KO mice, and monitored tumor growth, morphology, histology, and intra-tumoral microvessel density.ResultsAllografted melanoma tumors that developed in CIB1-KO mice were smaller in volume, had a distinct necrotic appearance, and had significantly less intra-tumoral microvessel density. Similarly, allografted Lewis lung carcinoma tumors in CIB1-KO mice were smaller in volume and mass, and appeared to have decreased perfusion. Intra-tumoral hemorrhage, necrosis, and perivascular fibrosis were also increased in tumors that developed in CIB1-KO mice.ConclusionsThese findings suggest that, in addition to its other functions, CIB1 plays a critical role in facilitating tumor growth and tumor-induced angiogenesis.

Platelet neuropeptide Y is critical for ischemic revascularization in mice

We previously reported that the sympathetic neurotransmitter neuropeptide Y (NPY) is potently angiogenic, primarily through its Y2 receptor, and that endogenous NPY is crucial for capillary angiogenesis in rodent hindlimb ischemia. Here we sought to identify the source of NPY responsible for revascularization and its mechanisms of action. At d 3, NPY–/– mice demonstrated delayed recovery of blood flow and limb function, consistent with impaired collateral conductance, while ischemic capillary angiogenesis was reduced (~70%) at d 14. This biphasic temporal response was confirmed by 2 peaks of NPY activation in rats: a transient early increase in neuronally derived plasma NPY and increase in platelet NPY during late‐phase recovery. Compared to NPY‐null platelets, collagen‐activated NPY‐rich platelets were more mitogenic (~2‐fold vs. ~1.6‐fold increase) for human microvascular endothelial cells, and Y2/Y5 receptor antagonists ablated this difference in proliferation. In NPY+/+ mice, ischemic angiogenesis was prevented by platelet depletion and then restored by transfusion of platelets from NPY+/+ mice, but not NPY–/– mice. In thrombocytopenic NPY–/– mice, transfusion of wild‐type platelets fully restored ischemia‐induced angiogenesis. These findings suggest that neuronally derived NPY accelerates the early response to femoral artery ligation by promoting collateral conductance, while platelet‐derived NPY is critical for sustained capillary angiogenesis.—Tilan, J. U., Everhart, L. M., Abe, K., Kuo‐Bonde, L., Chalothorn, D., Kitlinska, J., Burnett, M. S., Epstein, S. E., Faber, J. E., Zukowska, Z. Platelet neuropeptide Y is critical for ischemic revascularization in mice. FASEB J. 27, 2244–2255 (2013). www.fasebj.org