Angiogenesis is the formation of new blood vessels by sprouting from pre-existing ones. Transforming growth factor-beta (TGFβ) promotes angiogenesis and is a known inducer of endothelial-mesenchymal transition (EndMT), a process whereby endothelial cells become fibroblastic and motile. We hypothesize that TGFβ-induced EndMT enables endothelial cells to detach from the mature vessel and migrate to form the sprout that becomes a new vessel during angiogenesis. This study characterized EndMT in response to TGFβ +/- vascular endothelial growth factor (VEGF). Bovine aortic endothelial cells (BAEC) were stimulated with TGFβ +/- VEGF for prolonged periods. Confocal imaging and immunoblotting analyses revealed the strongest EndMT response at 5 ng/ml of TGFβ after 144 hours of exposure. A three-dimensional collagen model of angiogenesis revealed a potential relationship between EndMT and blood vessel sprouting. These results suggest that EndMT induction in BAECs requires high concentrations and prolonged exposure to TGFβ and is not significantly influenced by VEGF. / NSERC

Transforming growth factor-beta (TGFβ) is an important signaling molecule that regulates several cellular processes including angiogenesis. However, its effects on angiogenesis are complex, with it being pro-angiogenic only at low concentrations (Pepper et al., 1993). Evidence suggests that downstream signaling pathways of TGFβ may be activated in a dose-dependent fashion. In fact, previous work in our laboratory has shown that the non-canonical Par6 polarity pathway gets preferentially activated at low concentrations. Considering the different cellular effects of downstream signaling pathways, we propose that TGFβ may modulate its effects on angiogenesis via differential activation of the canonical Smad and non-canonical Akt, FAK, NFκB and Par6 polarity signaling pathways. Based on this premise, bovine aortic endothelial cells were treated with TGFβ1 and TGFβ2 at concentrations ranging from 0.05 to 5 ng/mL. The activation patterns of canonical and non-canonical signaling pathways were studied via western blotting; with the use of phospho-specific antibodies against Smad2, Akt, FAK and NFκB. Preliminary results reveal that high concentrations of both TGFβ1 and TGFβ2 (5 ng/mL) cause preferential activation of Smad2, while the Akt, FAK and NFκB signaling pathways do not appear to become activated in response to TGFβ1 or TGFβ2 at the concentrations and time points studied. These results suggest the effect of TGFβ on angiogenesis may not involve Akt, FAK or NFκB signaling, but may involve dose-dependent signaling of the Smad signaling pathway. / NSERC

Vascular endothelial cells (VECs) play an essential role in regulating the passage of macromolecules and cells between the blood stream and underlying tissues. The second messenger 3’, 5’ cyclic adenosine monophosphate (cAMP) regulates numerous events in VECs, including permeability. Since human VECs express several distinct cAMP-hydrolyzing phosphodiesterases (PDEs), and these are the only enzymes that catalyze the inactivation of cAMP, we investigated if selective pharmacological inhibition of PDEs could impact VEC permeability. Interestingly, we found that PDE4 inhibitors decreased human aortic VEC (HAEC) permeability and PDE4 and PDE3 inhibitors decreased human microvascular VEC (HMVEC) permeability. Consistent with a role for both protein kinase A (PKA) and exchange protein activated by cAMP (EPAC) in regulating VEC permeability, selective activators of these enzymes significantly decreased permeability. Since neither PDE4 nor PDE3 inhibitors significantly increased cAMP in these cells, our data are consistent with the idea that PDE inhibition causes small localized increases in “pools” of cAMP that regulate permeability. In order to test if PDE4 enzymes could act locally on pools of cAMP that regulated permeability, we selectively isolated the adherens junctional protein VE-cadherin from confluent monolayers of HAECs or HMVECs, and immunoblotted these isolates for cAMPeffectors and PDEs. Briefly, we found that each PKA-II, EPAC1, and a PDE4D variant, but not PDE3 enzymes, each could be isolated in VE-cadherin-based complexes from these cells. These novel findings identify PKA-II, EPAC1, and PDE4D as members of VE-cadherin-based signaling complexes in human VECs and are consistent with the idea that localized cAMP-signaling regulates permeability in these cells. / Thesis (Master, Pathology & Molecular Medicine) -- Queen's University, 2007-09-14 15:52:20.216

Multiple lines of evidence suggest that elevated plasma lipoprotein(a) (Lp(a)) concentrations are a significant risk factor for the development of a number of vascular diseases including coronary heart disease and stroke. Lp(a) consists of a low-density lipoprotein (LDL)-like moiety and an unique glycoprotein, apolipoprotein(a) (apo(a)), that is covalently attached to the apolipoproteinB-100 (apoB-100) component of LDL by a single disulfide bond. Many studies have suggested a role for Lp(a) in the process of endothelial dysfunction. Indeed, Lp(a) has been shown to increase both the expression of adhesion molecules on endothelial cells (EC), as well as monocyte and leukocyte chemotactic activity in these cells. We have previously demonstrated that Lp(a), through its apo(a) moiety, increases actomyosin-driven EC contraction which, as a consequence, increases EC permeability. In this thesis, we have demonstrated a role for the strong lysine-binding site in the kringle IV type 10 domain of apo(a) in increasing EC permeability, which occurs through a Rho/Rho kinase-dependent pathway. We have further validated these findings using mouse mesenteric arteries in a pressure myograph system. We also have dissected another major signaling pathway initiated by apo(a) that involves in a disruption of adherens junctions in EC. In this pathway, apo(a)/Lp(a) activates the PI3K/Akt/GSK3β-dependent pathway to facilitate nuclear translocation of beta-catenin. In the nucleus beta-catenin induced the expression of cyclooxygenase-2 (COX-2) and the secretion of prostaglandin E2 (PGE2) from the EC. Finally, we have presented data to suggest a novel inflammatory role for apo(a) in which it induces the activation of nuclear factor-kappaB through promotion of the dissociation of IkappaB from the inactive cytoplasmic complex; this allows the nuclear translocation of NFkappaB with attendant effects on the transcription of pro-inflammatory genes. Taken together, our findings may facilitate the development of new drug targets for mitigating the harmful effects of Lp(a) on vascular EC which corresponds to an early step in the process of atherogenesis. / Thesis (Ph.D, Biochemistry) -- Queen's University, 2009-09-22 19:24:04.594

Few studies have compared cortisol responses to psychological and physical stress tasks with or without social evaluation. Flow-mediated dilation (FMD) with reactive hyperemia is impaired following acute stress though less is known regarding the impact of stress and cortisol on FMD with exercise induced increases in shear stress (EX-FMD). The purpose of this study was 1) to compare cortisol responses between the Trier Social Stress Test (TSST) and 5min cold pressor test with and without social evaluation (CPT, CPT+SE) and 2) to examine the impact of these stressors and cortisol elevation on EX-FMD. 59 healthy male subjects were randomly assigned to one of three conditions: TSST, CPT, or CPT+SE. Brachial artery EX-FMD was assessed before, 15 and 35min post-stress with Echo and Doppler ultrasound. Results are mean ± SD. Baseline parameters did not differ between conditions (p>.05) and stress responses were similar between the three conditions for peak cortisol: TSST 11.34±5.53nmol/L; CPT 10.08±4.48nmol/L; CPT+SE 8.51±3.60nmol/L; condition effect p=.292; cortisol responders only (increase >2nmol/L) TSST 12.48±5.24nmol/L; CPT 12.13±5.31nmol/L; CPT+SE 10.70±2.86nmol/L; condition effect p=.560; peak MAP: 131.99 ±18.56mmHg, condition effect p=.664; peak TPR: 25.84 ± 9.78mmHg/L/min, condition effect p=0.841; peak stress rating out of 10: 5.11 ± 2.12, condition effect p= 0.292; with the exception of HR (TSST: 95.06 ± 15.29bpm; CPT: 79.00 ± 11.85bpm; CPT+SE: 77.98 ± 7.66bpm; condition effect p= 0.003) and pain ratings out of 10 (TSST: 1.21 ± 1.72; CPT: 6.66 ± 1.42; CPT+SE: 6.38 ± 1.73; condition effect p<.001). Shear stress was lower in the 15min post-stress vs. pre stress and 35min post-stress trials (72.34 ± 4.46; 15min post-stress: 70.79 ± 5.39; 35min post-stress: 70.60 ± 6.10; condition effect p= 0.592; trial effect: p= 0.018). EX-FMD increased from pre- stress to 15min post-stress in all conditions (pre-stress 6.22 ± 2.75%; 15min-post stress: 7.91 ± 3.24%; 35min post-stress: 6.60 ± 2.93%; trial effect p<.001). No correlation between change in EX-FMD and change in cortisol was detected (r2= 0.0125; p=.404). In conclusion, the TSST, CPT and CPT+SE elicited similar stress responses and stress transiently enhanced EX-FMD. Cortisol responses did not explain the enhanced EX-FMD post stress. / Thesis (Master, Kinesiology & Health Studies) -- Queen's University, 2012-09-14 15:41:33.325

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Previous work in our laboratory indicates that the diameter of brain arteries and arterioles can be increased by N-methyl-D-aspartate (NMDA) receptor activation. We looked for expression of NMDA receptors and endothelial cell responses to NMDA receptor agonists and antagonists in the mouse brain endothelial cell line bEnd.3. Using RT-PCR and Western blotting we found evidence supporting the presence of NMDA receptor subunits NR1 and NR2C. Treatment of bEnd.3 cells with combinations of 100 μM glutamate and D-serine significantly increased intracellular calcium. However, we saw no direct evidence that NO was produced in response to NMDA receptor activation using the Griess method. We did observe an NMDA receptor-dependent increase in protein nitrosylation. This increase is unlikely related to enhanced NO levels since it was not correlated with NO production and was not inhibited by the endothelial NO synthase inhibitor L-NIO.

Modular tissue engineering involves assembling tissue constructs with integral vasculature from units containing adipose-derived mesenchymal stromal cells (adMSCs) and endothelial cells. Here, the effects of implant volume and adMSC density on the vascularization of modular tissues were explored. Both parameters affected the contributions of host- and graft-derived vessels, without affecting total vessel density. Increasing implant volume from 0.01 to 0.10 mL increased HIF1&alpha; expression and graft-derived vessel density, suggesting a role of hypoxia in graft-derived vessel formation. However, increasing adMSC density within small-volume implants did not increase HIF1&alpha; expression. Vascularization of small-volume implants of high (4.3&bull;10^6 cells/mL) and low (1.0&bull;10^6 cells/mL) adMSC densities was dominated by host vessel ingrowth at day 7. By increasing adMSC density, a high proportion of host-derived vessels was maintained to day 14, presumably via paracrine effects. Further dissection of the role of hypoxia in modular tissue engineering remains a promising avenue to pursue.

Modular tissue engineering involves assembling tissue constructs with integral vasculature from units containing adipose-derived mesenchymal stromal cells (adMSCs) and endothelial cells. Here, the effects of implant volume and adMSC density on the vascularization of modular tissues were explored. Both parameters affected the contributions of host- and graft-derived vessels, without affecting total vessel density. Increasing implant volume from 0.01 to 0.10 mL increased HIF1&alpha; expression and graft-derived vessel density, suggesting a role of hypoxia in graft-derived vessel formation. However, increasing adMSC density within small-volume implants did not increase HIF1&alpha; expression. Vascularization of small-volume implants of high (4.3&bull;10^6 cells/mL) and low (1.0&bull;10^6 cells/mL) adMSC densities was dominated by host vessel ingrowth at day 7. By increasing adMSC density, a high proportion of host-derived vessels was maintained to day 14, presumably via paracrine effects. Further dissection of the role of hypoxia in modular tissue engineering remains a promising avenue to pursue.