Scott M. Moore

Congenic Fine-Mapping Identifies a Major Causal Locus for Variation in the Native Collateral Circulation and Ischemic Injury in Brain and Lower Extremity

Rationale: Severity of tissue injury in occlusive disease is dependent on the extent (number and diameter) of collateral vessels, which varies widely among healthy mice and humans. However, the causative genetic elements are unknown. Recently, much of the variation among different mouse strains, including C57Bl/6J (B6, high extent) and BALB/cByJ (Bc, low extent), was linked to a quantitative trait locus on chromosome 7 (Candq1). Objective: We used congenic mapping to refine Candq1 and its candidate genes to create an isogenic strain set with large differences in collateral extent to assess their impact and the impact of Candq1, alone, on ischemic injury. Methods and Results: Six congenic strains possessing portions of Candq1 introgressed from B6 into Bc were generated and phenotyped. Candq1 was refined from 27 to 0.737 Mb with full retention of effect, that is, return or rescue of phenotypes from the poor values in Bc to nearly those of wild-type B6 in the B6/B6 congenic mice as follows: 83% rescue of low pial collateral extent and 4.5-fold increase in blood flow and 85% reduction of infarct volume after middle cerebral artery occlusion; 54% rescue of low skeletal muscle collaterals and augmented recovery of perfusion (83%) and function after femoral artery ligation. Gene deletion and in silico analysis further delineated the candidate genes. Conclusions: We have significantly refined Candq1 (now designated determinant of collateral extent 1; Dce1), demonstrated that genetic background–dependent variation in collaterals is a major factor underlying differences in ischemic tissue injury, and generated a congenic strain set with wide allele dose–dependent variation in collateral extent for use in investigations of the collateral circulation.

Lysine Acetyltransferase PCAF Is a Key Regulator of Arteriogenesis

Objective—Therapeutic arteriogenesis, that is, expansive remodeling of preexisting collaterals, using single-action factor therapies has not been as successful as anticipated. Modulation of factors that act as a master switch for relevant gene programs may prove more effective. Transcriptional coactivator p300-CBP–associated factor (PCAF) has histone acetylating activity and promotes transcription of multiple inflammatory genes. Because arteriogenesis is an inflammation-driven process, we hypothesized that PCAF acts as multifactorial regulator of arteriogenesis. Approach and Results—After induction of hindlimb ischemia, blood flow recovery was impaired in both PCAF−/− mice and healthy wild-type mice treated with the pharmacological PCAF inhibitor Garcinol, demonstrating an important role for PCAF in arteriogenesis. PCAF deficiency reduced the in vitro inflammatory response in leukocytes and vascular cells involved in arteriogenesis. In vivo gene expression profiling revealed that PCAF deficiency results in differential expression of 3505 genes during arteriogenesis and, more specifically, in impaired induction of multiple proinflammatory genes. Additionally, recruitment from the bone marrow of inflammatory cells, in particular proinflammatory Ly6Chi monocytes, was severely impaired in PCAF−/− mice. Conclusions—These findings indicate that PCAF acts as master switch in the inflammatory processes required for effective arteriogenesis.

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.

A method for evaluating the murine pulmonary vasculature using micro-computed tomography

BACKGROUND Significant mortality and morbidity are associated with alterations in the pulmonary vasculature. While techniques have been described for quantitative morphometry of whole-lung arterial trees in larger animals, no methods have been described in mice. We report a method for the quantitative assessment of murine pulmonary arterial vasculature using high-resolution computed tomography scanning. METHODS Mice were harvested at 2 weeks, 4 weeks, and 3 months of age. The pulmonary artery vascular tree was pressure perfused to maximal dilation with a radio-opaque casting material with viscosity and pressure set to prevent capillary transit and venous filling. The lungs were fixed and scanned on a specimen computed tomography scanner at 8-μm resolution, and the vessels were segmented. Vessels were grouped into categories based on lumen diameter and branch generation. RESULTS Robust high-resolution segmentation was achieved, permitting detailed quantitation of pulmonary vascular morphometrics. As expected, postnatal lung development was associated with progressive increase in small-vessel number and arterial branching complexity. CONCLUSIONS These methods for quantitative analysis of the pulmonary vasculature in postnatal and adult mice provide a useful tool for the evaluation of mouse models of disease that affect the pulmonary vasculature.