Objectives— Ephrin ligands and Eph receptors are signaling molecules that are differentially expressed on arteries and veins during development. We examined whether Eph-B4, a venous marker, and Ephrin-B2, an arterial marker, are regulated during vein graft adaptation in humans and aged rats. Methods and Results— Eph-B4 transcripts and immunodetectable protein are downregulated in endothelial and smooth muscle cells of patent vein grafts in both humans and in aged rats, whereas Ephrin-B2 transcripts and protein are not strongly induced. Other markers of arterial identity, including dll4 and notch-4, are also not induced during vein graft adaptation in aged rats. Because VEGF-A is upstream of the Ephrin–Eph pathway, and expression of VEGF-A is induced only at early time points after exposure of the vein to the arterial environment, we inhibited VEGF-A in vein grafts using an siRNA-based approach. Vein grafts treated with siRNA directed against VEGF-A demonstrated a thicker intima-media containing α-actin, consistent with arterialization, but did not contain Eph-B4 or Ephrin-B2. Conclusions— Venous identity is preserved in the veins of aged animals, but is lost during adaptation to the arterial circulation; arterial markers are not induced. Markers of vessel identity are plastic in adults and their selective regulation may mediate vein graft adaptation to the arterial environment in aged animals and humans.

In this study, preoperative CA 19-9 values were strongly associated with the identification of subradiographic unresectable disease. Preoperative CA 19-9 values may allow surgeons to better select patients for staging laparoscopy.

Vascular smooth muscle cells exhibit varied responses after vessel injury and surgical interventions, including phenotypic switching, migration, proliferation, protein synthesis, and apoptosis. Although the source of the smooth muscle cells that accumulate in the vascular wall is controversial, possibly reflecting migration from the adventitia, from the circulating blood, or in situ differentiation, the intracellular signal transduction pathways that control these processes are being defined. Some of these pathways include the Ras-mitogen-activated protein kinase, phosphatidylinositol 3-kinase-Akt, Rho, death receptor-caspase, and nitric oxide pathways. Signal transduction pathways provide amplification, redundancy, and control points within the cell and culminate in biologic responses. We review some of the signaling pathways activated within smooth muscle cells that contribute to smooth muscle cell heterogeneity and development of pathology such as restenosis and neointimal hyperplasia.

Objective(s) Older patients are thought to tolerate acute ischemia more poorly than younger patients. Since aging may depress both angiogenesis and arteriogenesis, we determined the effects of age on both angiogenesis and arteriogenesis, in a model of severe acute limb ischemia. Methods Young adult (3 month) and aged (18 month) C57BL/6 mice underwent right common iliac artery and vein ligation and transection. Data were collected on days 0, 7, and 14. Perfusion was measured with laser Doppler and compared to the contralateral limb. Functional deficits were evaluated with the Tarlov scale. Capillary density and endothelial progenitor cell (EPC) number were determined by direct counting lectin-positive/alpha-actin-negative cells and VEGFR2/CXCR4 dually-positive cells, respectively; angiography was performed to directly assess arteriogenesis. Results Young adult and aged mice had a similar degree of decreased perfusion after iliac ligation (young, n=15: 20.4±1.9%, vs. old, n=20: 19.6±1.3%; p=.72, ANOVA); however, young mice recovered faster and to a greater degree than aged mice (day 7, 35±6% vs. 17±4%, p=.046; day 14, 60±5% vs. 27±7%, p=.0014). Aged mice had worse functional recovery by day 14 compared to young mice (2.3±.3 vs. 4.3±.4; p=.0021). Aged mice had increased capillary density (day 7, 12.9±4.4 vs. 2.8±0.3 capillaries/hpf; p=.02) and increased number of EPC incorporated into the ischemic muscle (day 7, 8.1±0.9 vs. 2.5±1.9 cells; p=0.007) compared to young mice, but diminished numbers of collateral vessels to the ischemic limb (1 vs. 9; p=0.01), as seen on angiography. Conclusions After severe hindlimb ischemia, aged animals become ischemic to a similar degree as young animals, but aged animals have significantly impaired arteriogenesis and functional recovery compared to younger animals. These results suggest that strategies to stimulate arteriogenesis may complement those that increase angiogenesis, and may result in improved relief of ischemia. Clinical Relevance The incidence of chronic limb ischemia increases with age as do the consequences of acute ischemia. We show, using a new model of severe acute limb ischemia that does not wound the ischemic limb, that aged mice increase angiogenesis in response to acute ischemia, but do not show arteriogenesis, i.e. large collateral formation. These results suggest why elderly patients develop large vessel disease such as claudication but can still heal small wounds. They also suggest that strategies to treat ischemia in elderly patients should focus on stimulating large vessel arteriogenesis, rather than solely small vessel angiogenesis.

Vascular smooth muscle cells (SMC) may be directly exposed to blood flow after an endothelial-denuding injury. It is not known whether direct exposure of SMC to shear stress reduces SMC turnover and contributes to the low rate of restenosis after most vascular interventions. This study examines if laminar shear stress inhibits SMC proliferation or stimulates apoptosis. Bovine aortic SMC were exposed to arterial magnitudes of laminar shear stress (11 dynes/cm(2)) for up to 24 h and compared to control SMC (0 dynes/cm(2)). SMC density was assessed by cell counting, DNA synthesis by (3)[H]-thymidine incorporation, and apoptosis by TUNEL staining. Akt, caspase, bax, and bcl-2 phosphorylation were assessed by Western blotting; caspase activity was also measured with an in vitro assay. Analysis of variance was used to compare groups. SMC exposed to laminar shear stress had a 38% decrease in cell number (n = 4, P = 0.03), 54% reduction in (3)[H]-thymidine incorporation (n = 3, P = 0.003), and 15-fold increase in TUNEL staining (n = 4, P < 0.0001). Akt phosphorylation was reduced by 67% (n = 3, P < 0.0001), whereas bax/bcl-2 phosphorylation was increased by 1.8-fold (n = 3, P = 0.01). Caspase-3 activity was increased threefold (n = 5, P = 0.03). Pretreatment of cells with ZVAD-fmk or wortmannin resulted in 42% increased cell retention (n = 3, P < 0.01) and a fourfold increase in apoptosis (n = 3, P < 0.04), respectively. Cells transduced with constitutively-active Akt had twofold decreased apoptosis (n = 3, P < 0.002). SMC exposed to laminar shear stress have decreased proliferation and increased apoptosis, mediated by the Akt pathway. These results suggest that augmentation of SMC apoptosis may be an alternative strategy to inhibit restenosis after vascular injury.

Both neointimal hyperplasia and inward remodeling contribute to restenosis and lumen loss. Nogo-B has been recently described as an inhibitor of vascular injury and neointimal hyperplasia. To determine whether Nogo-B expression may be a mediator of inward remodeling, we examine the localization of expression of Nogo-B in an in vivo model that examines both neointimal hyperplasia and inward remodeling. The rabbit carotid artery was subjected to balloon injury, outflow branch ligation to reduce flow, or both balloon injury and reduction in flow. In balloon injury-induced neointimal hyperplasia Nogo-B expression was reduced in the intima and media but stimulated in the adventitia. In low flow-induced inward remodeling medial Nogo-B expression was not reduced and adventitial Nogo-B expression was not stimulated. Low flow significantly augmented balloon injury-induced neointimal hyperplasia and was accompanied by reduced intimal and medial Nogo-B expression, and increased adventitial Nogo-B expression in both smooth muscle cells and macrophages. Low flow-induced inward remodeling is not associated with changes in medial Nogo-B expression and is distinct from injury-induced neointimal hyperplasia. Pharmacological strategies to inhibit neointimal hyperplasia and restenosis using normal flow models may only partially account for lumen loss and therefore may not accurately predict responses in patients with extensive outflow disease.

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