Stephen P. Maloney

Venous Identity Is Lost but Arterial Identity Is Not Gained During Vein Graft Adaptation

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.

Limb ischemia after iliac ligation in aged mice stimulates angiogenesis without arteriogenesis

OBJECTIVE 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-months-old) and aged (18-months-old) 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 a laser Doppler scan 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 aged, n = 20: 19.6 +/- 1.3%; P = .72, analysis of variance [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 +/- 0.3 vs 4.3 +/- 0.4; 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 = .007) compared to young mice, but diminished numbers of collateral vessels to the ischemic limb (1 vs 9; P = .01), as seen on angiography. CONCLUSION After severe hind limb 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.

Laminar shear stress stimulates vascular smooth muscle cell apoptosis via the Akt pathway.

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/cm2) for up to 24 h and compared to control SMC (0 dynes/cm2). 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. J. Cell. Physiol. 216: 389–395, 2008.

Endothelial Nitric Oxide Synthase Stimulates Aneurysm Growth in Aged Mice

Background/Aims: Age-associated changes in endothelial nitric oxide synthase (eNOS) expression have not been definitively linked to the pathophysiology of aortic aneurysms. We examined the role of eNOS in human patients and an age-appropriate mouse model. Methods: eNOS transcripts and immunodetectable protein were assessed by quantitative PCR and immunohistochemistry in human ascending thoracic aneurysms (n = 29) and referent aortae (n = 31). Carotid aneurysms were induced with CaCl2 in young adult (3 months) and aged (18 months) C57BL/6 and eNOS-knockout (eNOS-KO) mice. Results: eNOS transcripts and protein were reduced in human aneurysms compared with controls, although aortic eNOS expression also decreased with patient age. Aged wild-type mice had significantly larger aneurysm diameter than young adult mice. Aged wild-type mice had reduced eNOS transcripts and protein compared with young adult mice. Aged eNOS-KO mice had smaller aneurysms compared with aged wild-type mice but similar size aneurysms compared with young eNOS-KO and young wild-type mice. Conclusion: eNOS expression is reduced in both aged human and aged mouse endothelium and eNOS expression is linked to aneurysm expansion in aged but not young adult mice. These findings support the relevance of age-associated changes in eNOS expression in clinical aneurysmal disease.

Unusual manifestations of Clostridium difficile infection.

BACKGROUND Clostridium difficile infection (CDI) is an increasing nosocomial problem. New, more-virulent strains of C. difficile have spread across North America and Europe. Health care institutions now face a greater incidence of disease, often with greater severity. A need for surgical management for control of infection is on the increase. The clinical appearance of CDI is changed. METHODS We report four unusual and severe cases of CDI in surgical patients with a review of the relevant literature. RESULTS One patient developed CDI and required a colectomy for a perforated viscus. He developed C. difficile ileitis 12 days later that responded to medical therapy. Another patient who underwent a colectomy for infrarenal aortic occlusion, later in his hospital course, developed C. difficile ileitis and died. The third patient was hospitalized for several months for hypertension and associated morbidities. Eventually he developed severe abdominal pain and was found to have a small bowel mural abscess that grew C. difficile on culture. A fourth patient, taking long-term antibiotics for a surgical site infection of the knee, developed unexplained leukocytosis without diarrhea. Colonoscopy revealed pseudomembranous colitis that advanced to toxic megacolon. She required a colectomy and ultimately died from the disease. CONCLUSIONS Patients are at high risk from CDI in this modern era. Disease manifestations may differ from the typical presentation. A heightened awareness for diagnosing this dangerous, evolving disease is paramount.

Adjunctive pharmacologic use in carotid endarterectomy: a review.

Although carotid endarterectomy (CEA) is now widely accepted as the surgical therapy for carotid stenosis, the role of and indications and evidence for many pharmacologic agents that are used adjunctively in the perioperative setting have not been conclusively established. Aspirin (acetylsalicylic acid) is the pharmaceutical agent that has been studied most extensively in conjunction with CEA; other than aspirin and dextran, the use of many agents before, during, and after CEA has not been standardized. Prospective randomized trials are still needed to demonstrate efficacy, predict outcome, and determine the optimal use of these medications in their adjunctive use during CEA to improve patient care and obtain optimal surgical outcomes.

Murine carotid aneurysms develop without down regulation of Nogo-B expression: A novel model for human thoracic aneurysms

INTRODUCTION: Although abdominal aortic aneurysms are associated with smooth muscle cell (SMC) apoptosis, ascending thoracic aneurysms are associated with preserved SMC density. Decreased expression of Nogo-B has recently been described as a marker of vascular injury. To determine whether proximal thoracic aneurysms are associated with injury, we examined the expression of Nogo-B in human specimens and a mouse model.