Theresa W. Fossum

Upregulation of Vascular Arginase in Hypertension Decreases Nitric Oxide–Mediated Dilation of Coronary Arterioles

One characteristic of hypertension is a decreased endothelium-dependent nitric oxide (NO)-mediated vasodilation; however, the underlying mechanism is complex. In endothelial cells (ECs), l-arginine is the substrate for both NO synthase (NOS) and arginase. Because arginase has recently been shown to modulate NO-mediated dilation of coronary arterioles by reducing l-arginine availability, we hypothesized that upregulation of vascular arginase in hypertension contributes to decreased NO-mediated vasodilation. To test this hypothesis, hypertension (mean arterial blood pressure >150 mm Hg) was maintained for 8 weeks in pigs by aortic coarctation. Coronary arterioles from normotensive (NT) and hypertensive (HT) pigs were isolated and pressurized for in vitro study. NT vessels dilated dose-dependently to adenosine (partially mediated by endothelial release of NO) and sodium nitroprusside (endothelium-independent vasodilator). Conversely, HT vessels exhibited reduced dilation to adenosine but dilated normally to sodium nitroprusside. Adenosine-stimulated NO release was increased ≈3-fold in NT vessels but was reduced in HT vessels. Moreover, arginase activity was 2-fold higher in HT vessels. Inhibition of arginase activity by N&ohgr;-hydroxy-nor-l-arginine or incubation with l-arginine partially restored NO release and dilation to adenosine in HT vessels. Immunohistochemistry showed that arginase expression was increased but NOS expression was decreased in arteriolar ECs of HT vessels. These results suggest that NO-mediated dilation of coronary arterioles is inhibited in hypertension by an increase in arginase activity in EC, which limits l-arginine availability to NOS for NO production. The inability of arginase blockade or l-arginine supplementation to completely restore vasodilation may be related to downregulation of endothelial NOS expression.

Upregulation of Arginase by H2O2 Impairs Endothelium-Dependent Nitric Oxide-Mediated Dilation of Coronary Arterioles

Objective—Overproduction of reactive oxygen species such as hydrogen peroxide (H2O2) has been implicated in various cardiovascular diseases. However, mechanism(s) underlying coronary vascular dysfunction induced by H2O2 is unclear. We studied the effect of H2O2 on dilation of coronary arterioles to endothelium-dependent and endothelium-independent agonists. Methods and Results—Porcine coronary arterioles were isolated and pressurized without flow for in vitro study. All vessels developed basal tone and dilated dose-dependently to activators of nitric oxide (NO) synthase (adenosine and ionomycin), cyclooxygenase (arachidonic acid), and cytochrome P450 monooxygenase (bradykinin). Intraluminal incubation of vessels with H2O2 (100 &mgr;mol/L, 60 minutes) did not alter basal tone but inhibited vasodilations to adenosine and ionomycin in a manner similar as that by NO synthase inhibitor L-NAME. H2O2 affected neither endothelium-dependent responses to arachidonic acid and bradykinin nor endothelium-independent dilation to sodium nitroprusside. The inhibited adenosine response was not reversed by removal of H2O2 but was restored by excess L-arginine. Inhibition of L-arginine consuming enzyme arginase by α-difluoromethylornithine or N&ohgr;-hydroxy-nor-l-arginine also restored vasodilation. Administering deferoxamine, an inhibitor of hydroxyl radical production, prevented the H2O2-induced impairment of vasodilation to adenosine. Western blot and reverse-transcription polymerase chain reaction results indicated that arginase I was upregulated after treating vessels with H2O2. Conclusions—H2O2 specifically impairs endothelium-dependent NO-mediated dilation of coronary microvessels by reducing L-arginine availability through upregulation of arginase. The formation of hydroxyl radicals from H2O2 may contribute to this process.

Multilayer vascular grafts based on collagen-mimetic proteins.

A major roadblock in the development of an off-the-shelf, small-caliber vascular graft is achieving rapid endothelialization of the conduit while minimizing the risk of thrombosis, intimal hyperplasia, and mechanical failure. To address this need, a collagen-mimetic protein derived from group A Streptococcus, Scl2.28 (Scl2), was conjugated into a poly(ethylene glycol) (PEG) hydrogel to generate bioactive hydrogels that bind to endothelial cells (ECs) and resist platelet adhesion. The PEG-Scl2 hydrogel was then reinforced with an electrospun polyurethane mesh to achieve suitable biomechanical properties. In the current study, initial evaluation of this multilayer design as a potential off-the-shelf graft was conducted. First, electrospinning parameters were varied to achieve composite burst pressure, compliance, and suture retention strength that matched reported values of saphenous vein autografts. Composite stability following drying, sterilization, and physiological conditioning under pulsatile flow was then demonstrated. Scl2 bioactivity was also maintained after drying and sterilization as indicated by EC adhesion and spreading. Evaluation of platelet adhesion, aggregation, and activation indicated that PEG-Scl2 hydrogels had minimal platelet interactions and thus appear to provide a thromboresistant blood contacting layer. Finally, evaluation of EC migration speed demonstrated that PEG-Scl2 hydrogels promoted higher migration speeds than PEG-collagen analogs and that migration speed was readily tuned by altering protein concentration. Collectively, these results indicate that this multilayer design warrants further investigation and may have the potential to improve on current synthetic options.

Learning basic surgical skills with mental imagery: using the simulation centre in the mind

Context  Although surgeons and athletes frequently use mental imagery in preparing to perform, mental imagery has not been extensively researched as a learning technique in medical education.

Time Courses of Growth and Remodeling of Porcine Aortic Media During Hypertension: A Quantitative Immunohistochemical Examination

Arteries undergo marked structural and functional changes in human and experimental hypertension that generally involve smooth muscle cell (SMC) hypertrophy/hyperplasia as well as abnormal extracellular matrix turnover. In this study we examined time courses of changes in SMC activity and matrix protein content in a novel mini-pig aortic coarctation model. Cell proliferation was evaluated by immunostaining of Ki-67, apoptosis was assessed by TUNEL, and phenotypic changes were monitored by immunostaining three SMC contractile markers (caldesmon, calponin, and smoothelin). Changes in medial collagen and elastin were examined by picrosirius red and Verhoeff–van Gieson staining, respectively. LabVIEW-based image analysis routines were developed to objectively and efficiently quantify the (immuno)histochemical results. We found that significant cell proliferation and matrix production occurred in the early stages of this coarctation model and then declined gradually; the SMCs also tended to exhibit a less contractile phenotype following these cellular and extracellular changes. Specifically, different aspects of the phenotypic changes associated with hypertension occurred at different rates: cell proliferation and collagen production occurred early and peaked by 2 weeks, whereas changes in contractile protein expression continued to decrease over the entire 8-week study period. Temporal changes found in this study emphasize the importance of simultaneously tracing time courses of SMC growth and differentiation as well as matrix protein production and content. SMCs are multifunctional, and caution must be used to not overdefine phenotype. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.

In vivo response to an implanted shape memory polyurethane foam in a porcine aneurysm model

Cerebral aneurysms treated by traditional endovascular methods using platinum coils have a tendency to be unstable, either due to chronic inflammation, compaction of coils, or growth of the aneurysm. We propose to use alternate filling methods for the treatment of intracranial aneurysms using polyurethane-based shape memory polymer (SMP) foams. SMP polyurethane foams were surgically implanted in a porcine aneurysm model to determine biocompatibility, localized thrombogenicity, and their ability to serve as a stable filler material within an aneurysm. The degree of healing was evaluated via gross observation, histopathology, and low vacuum scanning electron microscopy imaging after 0, 30, and 90 days. Clotting was initiated within the SMP foam at time 0 (<1 h exposure to blood before euthanization), partial healing was observed at 30 days, and almost complete healing had occurred at 90 days in vivo, with minimal inflammatory response.

Transforming growth factor-β signaling in hypertensive remodeling of porcine aorta

A porcine aortic coarctation model was used to examine regulation of gene expression in early hypertensive vascular remodeling. Aortic segments were collected proximal (high pressure) and distal (low pressure) to the coarctation after 2 wk of sustained hypertension (mean arterial pressure>150 mmHg). Porcine 10K oligoarrays used for gene expression profiling of the two regions of aorta revealed downregulation of cytoskeletal and upregulation of extracellular region genes relative to the whole genome. A genomic database search for transforming growth factor-beta (TGF-beta) control elements showed that 19% of the genes that changed expression due to hypertension contained putative TGF-beta control elements. Real-time RT-PCR and microarray analysis showed no change in expression of TGF-beta1, TGF-beta2, TGF-beta3, or bone morphogenetic proteins-2 and -4, yet immunohistochemical staining for phosphorylated SMAD2, an indicator of TGF-beta signaling, and for phosphorylated SMAD1/5/8, an indicator of signaling through the bone morphogenetic proteins, showed the highest percentage of positively stained cells in the proximal aortic segments of occluded animals. For TGF-beta signaling, this increase was significantly different than for sham-operated controls. Western blot analysis showed no difference in total TGF-beta1 protein levels with respect to treatment or aortic segment. Immunohistochemistry showed that the protein levels of latency-associated peptide was decreased in proximal segments of occluded animals. Collectively, these results suggest that activation of TGF-beta, but not altered expression, may be a major mechanism regulating early hypertensive vascular remodeling.

Gastric and intestinal surgery.

Gastric surgery is commonly performed to remove foreign bodies and correct gastric dilatation-volvulus and is less commonly performed to treat gastric ulceration or erosion, neoplasia, and benign gastric outflow obstruction. Intestinal surgery, although commonly performed by veterinarians, should never be considered routine. The most common procedures of the small intestinal tract performed in dogs and cats include enterotomy and resection/anastomosis. Surgery of the large intestine is indicated for lesions causing obstruction, perforations, colonic inertia, or chronic inflammation.

Video-assisted left partial arytenoidectomy by diode laser photoablation for treatment of canine laryngeal paralysis.

OBJECTIVES To evaluate the clinical outcome of left partial arytenoidectomy by video-assisted laser diode photoablation as a surgical treatment for canine laryngeal paralysis (LP). STUDY DESIGN Case series. ANIMALS Dogs with bilateral LP (n=20). METHODS After endoscopic diagnosis of bilateral LP, left partial arytenoidectomy was performed by photoablation of arytenoid cartilage tissue using a diode laser (600 microm diameter, 15 W power, 980 nm wave length) to increase the width of the rima glottidis. Outcome was evaluated endoscopically (1 and 6 months) and clinically (1, 6, and 12 months). RESULTS No substantial complications occurred during photoablation or in the immediate postoperative period. Postoperative width of the rima glottidis ranged from 6 to 10 mm at its widest aspect. At 1 month, respiratory function after walking and short running appeared good. Clinical and endoscopic examination revealed good outcome at 1 and 6 months. At 6 months, there was no evidence of hypertrophic scar, hypertrophic granulation tissue, or stricture of the laryngeal glottis in any dog. Two dogs developed aspiration pneumonia after 12 months. CONCLUSIONS Partial arytenoidectomy using video-assisted diode laser photoablation appears to be an effective technique for treating LP. CLINICAL RELEVANCE Partial arytenoidectomy by diode laser photoablation should be considered as an alternative technique for treatment of canine LP.

TP508 (Chrysalin®) Reverses Endothelial Dysfunction and Increases Perfusion and Myocardial Function in Hearts With Chronic Ischemia

Endothelial dysfunction (ED) is characterized by impaired nitric oxide (NO) signaling, decreased NO-dependent vasodilatation, increased vascular inflammation, and diminished response to angiogenic factors. TP508 (Chrysalin®), an angiogenic tissue repair peptide, was tested for potential effects on myocardial revascularization and ED using a porcine model of chronic myocardial ischemia. TP508 increased perfusion in ischemic regions up to16-fold (P < .02) and doubled myocardial wall thickening (P < .02) relative to placebo controls. Ischemic arterioles exhibited impaired NO-mediated vasodilation and diminished NO production. TP508 reversed ischemic effects, increasing NO-mediated vasodilation (P < .05), endothelial nitric oxide synthase (eNOS) expression, and NO production. In human endothelial cells, TP508 stimulated eNOS activation (1.84 ± 0.2-fold; P < .02), increased NO production (85 ± 18%; P < .02), and prevented hypoxia-induced eNOS downregulation (P < .01). Thus, TP508 reverses ED both in porcine ischemic hearts and cultured human endothelial cells. These results suggest potential therapeutic benefit of TP508 in myocardial revascularization and treatment of ED-related diseases.