Tamara N. Fitzgerald

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.

Defining Surgical Therapy for Pseudomembranous Colitis With Toxic Megacolon

Background Pseudomembranous colitis has increased in incidence and severity over the past 10 years. Toxic megacolon is a rare but reported presentation of severe pseudomembranous colitis. This article reviews the reported cases of Clostridium difficile with toxic megacolon in the literature and introduces an additional case that underscores the importance of early diagnosis in guiding appropriate therapy. Methods A systematic review of the literature was performed to identify previous reports of pseudomembranous colitis presenting with toxic megacolon, and the outcomes of each of these cases was analyzed. The review was focused on atypical presentations in immunocompromised patients. Results Seventeen cases of C. difficile colitis presenting as toxic megacolon were identified. The overall mortality rate was 50% (9/18). Fifteen patients underwent surgery with an associated mortality rate of 50%. Thirteen patients had a subtotal colectomy. Seven of the patients (39%) were taking immunosuppressant medications, and 5 (28%) patients presented with atypical symptoms. Three (76%) of those were immunosuppressed. In several cases, failure to make an early diagnosis of C. difficile colitis resulted in a worse outcome because appropriate therapy was delayed. Conclusions Toxic megacolon is well-established as an unusual presentation of C. difficile colitis. These patients are less likely to present with typical symptoms such as diarrhea or typical risk factors like recent administration of antibiotics, so diagnosis can be a challenge. A patient presenting with toxic megacolon without a history of inflammatory bowel disease should be assumed to have C. difficile colitis until proven otherwise, and medical or surgical therapy administered accordingly.

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.

Computer-Based Endoscopy Simulation: Emerging Roles in Teaching and Professional Skills Assessment

Advances in endoscopy simulation are reviewed with emphasis on applications in teaching and skills assessment. Endoscopy simulation has only been realized recently in a computer-based fashion because of advances in technology, but several studies have been performed both to validate computer-based endoscopy simulators and to assess their potential role in training. Multiple studies have shown that simulators can distinguish between clinicians at different skill levels and also have shown improvement in clinician skill, particularly at the early stages of training. This article summarizes those studies. The cost versus benefit of endoscopic simulators is also discussed, as well as the upcoming role of simulators in judging competence and as a tool in the credentialing process.

Control of Blood Vessel Identity: From Embryo to Adult

Arteries and veins have been historically defined by the direction of blood flow and oxygen tension within the vessel, in addition to their functional, hemodynamic, and anatomical differences. It is now known that the molecular identity of these vessels is genetically predetermined, with specific molecular pathways activated during the development of arteries and veins. Eph-B4 is a determinant of venous differentiation and Ephrin-B2 is a determinant of arterial differentiation. Placement of a vein into the higher pressure and flow of the arterial circulation results in adaptation of the vein to the arterial environment. There is selective loss of Eph-B4 expression without induction of Ephrin-B2 expression during vein graft adaptation. These findings suggest that loss of venous identity is the crucial mechanism in vein graft adaptation and that developmentally critical determinants of vessel identity are plastic during adult life.

Diminished omega-3 fatty acids are associated with carotid plaques from neurologically symptomatic patients: Implications for carotid interventions.

The omega-3 fatty acids, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), are prevalent in fish oil and their cardioprotective effects are thought to be mediated by anti-inflammatory mechanisms. The aim of this study is to determine whether omega-3 fatty acids are associated with carotid plaques from neurologically symptomatic patients. Plaques were obtained from 41 patients (mean age 62 [44-84]; 24-asymptomatic, 17-symptomatic). Intra-plaque lipids were assessed with mass spectrometry. Compared to asymptomatic patients, significantly diminished omega-3 fatty acids DHA (545.8+/-98 ng/g vs. 270.7+/-19.6 ng/g, p=0.0096) and EPA (385.9+/-68 ng/g vs. 216.4+/-17.6 ng/g, p=0.0189) were found in carotid plaques from neurologically symptomatic patients. However, no differences were found in the levels of the omega-6 fatty acid arachidonic acid (p=0.2003). Immunohistochemistry and ELISA analysis (CD68(+) cells, 0.461+/-0.04 vs. 0.312+/-0.03, p=0.003) demonstrated an increased inflammatory infiltrate in plaques from neurologically symptomatic, compared to asymptomatic, patients. Carotid plaques from neurologically symptomatic patients are inflammatory and have decreased intra-plaque levels of omega-3 fatty acids. Future trials will determine whether interventions that increase omega-3 fatty acid incorporation into carotid plaques prevent stroke and improve the safety of carotid interventions.

Endovascular aneurysm repair is associated with less malnutrition than open abdominal aortic aneurysm repair

BACKGROUND Patients undergoing abdominal aortic aneurysm (AAA) repair have high rates of postoperative malnutrition. We examined whether endovascular aneurysm repair (EVAR) is associated with reduced postoperative malnutrition compared with open AAA repair. METHODS The records of patients undergoing AAA repair in the Veterans Affairs (VA) Connecticut Healthcare System were reviewed. Primary outcomes were 30-day morbidity, lengths of hospitalization and intensive care unit stay, duration of intubation, and nutritional risk index scores. RESULTS Sixty-two patients were included (open repair, 37; EVAR, 25). Nutritional parameters were comparable between groups before surgery. Patients treated with EVAR had improved postoperative nutritional profiles as determined by albumin level (3.7 +/- .08 vs 3.2 +/- .12; P = .003), and nutritional risk index (97.9 +/- 1.3 vs 88.9 +/- 1.8; P = .0006), compared with patients treated with open repair. CONCLUSIONS Patients undergoing EVAR developed significantly less postoperative malnutrition compared with those having open repair. EVAR may be a strategy to avoid malnutrition and improve outcomes in patients at risk for malnutrition after undergoing AAA repair.

Elevated Monocytes in Patients with Critical Limb Ischemia Diminish After Bypass Surgery

BACKGROUND Mononuclear cells (MNC) increase neovascularization and ulcer healing after injection into an ischemic extremity. Circulating MNC are composed of lymphocytes (85%), monocytes (15%), and endothelial progenitor cells (EPC; 0.03%). We hypothesized that ischemic limbs secrete paracrine signals to recruit bone marrow-derived monocytes and EPC into the circulation, such that patients with critical limb ischemia (CLI) have increased circulating monocytes compared with control patients. We also hypothesized that circulating monocytes and EPC recruitment decrease after resolution of ischemia with successful revascularization. METHODS We reviewed the records of all patients at the VA Connecticut Healthcare System undergoing primary, functionally successful, lower extremity peripheral bypass surgery between 2002 and 2007, but only including patients with both preoperative and postoperative (>4 mo) complete blood counts with differentials. RESULTS Patients with CLI (n = 24) had elevated preoperative monocyte counts compared with control patients (n = 8) (0.753 ± 0.04 versus 0.516 ± 0.05; P = 0.0046), whereas the preoperative lymphocyte counts were not significantly different. After revascularization, ischemic patients had decreased monocyte counts compared with control patients (-20% versus + 55%; P = 0.0003), although lymphocyte counts were unchanged in both groups. Diabetic patients also had reduced postoperative monocyte counts (-32% versus + 13%; P = 0.035). Multivariable logistic regression demonstrated that the only factor that independently predicted reduced postoperative monocyte count was preoperative CLI (P = 0.038). CONCLUSIONS Patients with CLI have increased numbers of circulating monocytes, and the monocyte number decreases with resolution of ischemia after successful revascularization. Circulating monocytes may be a clinically useful perioperative marker in patients with CLI undergoing vascular surgery.