Muneera R. Kapadia

Nitric oxide and nanotechnology: A novel approach to inhibit neointimal hyperplasia

OBJECTIVE Nitric oxide (NO) has been shown to inhibit neointimal hyperplasia after arterial interventions in several animal models. To date, however, NO-based therapies have not been used in the clinical arena. Our objective was to combine nanofiber delivery vehicles with NO chemistry to create a novel, more potent NO-releasing therapy that can be used clinically. Thus, the aim of this study was to evaluate the perivascular application of spontaneously self-assembling NO-releasing nanofiber gels. Our hypothesis was that this application would prevent neointimal hyperplasia. METHODS Gels consisted of a peptide amphiphile, heparin, and a diazeniumdiolate NO donor (1-[N-(3-Aminopropyl)-N-(3-ammoniopropyl)]diazen-1-ium-1,2-diolate [DPTA/NO] or disodium 1-[(2-Carboxylato)pyrrolidin-1-yl]diazen-1-ium-1,2-diolate [PROLI/NO]). Nitric oxide release from the gels was evaluated by the Griess reaction, and scanning electron microscopy confirmed nanofiber formation. Vascular smooth muscle cell (VSMC) proliferation and cell death were assessed in vitro by (3)H-thymidine incorporation and Personal Cell Analysis (PCA) system (Guava Technologies, Hayward, Calif). For the in vivo work, gels were modified by reducing the free-water content. Neointimal hyperplasia after periadventitial gel application was evaluated using the rat carotid artery injury model at 14 days (n = 6 per group). Inflammation and proliferation were examined in vivo with immunofluorescent staining against CD45, ED1, and Ki67 at 3 days (n = 2 per group), and graded by blinded observers. Endothelialization was assessed by Evans blue injection at 7 days (n = 3 per group). RESULTS Both DPTA/NO and PROLI/NO, combined with the peptide amphiphile and heparin, formed nanofiber gels and released NO for 4 days. In vitro, DPTA/NO inhibited VSMC proliferation and induced cell death to a greater extent than PROLI/NO. However, the DPTA/NO nanofiber gel only reduced neointimal hyperplasia by 45% (intima/media [I/M] area ratio, 0.45 +/- 0.07), whereas the PROLI/NO nanofiber gel reduced neointimal hyperplasia by 77% (I/M area ratio, 0.19 +/- 0.03, P < .05) vs control (injury alone I/M area ratio, 0.83 +/- 0.07; P < .05). Both DPTA/NO and PROLI/NO nanofiber gels significantly inhibited proliferation in vivo (1.06 +/- 0.30 and 0.19 +/- 0.11 vs injury alone, 2.02 +/- 0.20, P < .05), yet had minimal effect on apoptosis. Only the PROLI/NO nanofiber gel inhibited inflammation (monocytes and leukocytes). Both NO-releasing nanofiber gels stimulated re-endothelialization. CONCLUSIONS Perivascular application of NO-releasing self-assembling nanofiber gels is an effective and simple therapy to prevent neointimal hyperplasia after arterial injury. Our study demonstrates that the PROLI/NO nanofiber gel most effectively prevented neointimal hyperplasia and resulted in less inflammation than the DPTA/NO nanofiber gel. This therapy has great clinical potential to prevent neointimal hyperplasia after open vascular interventions in patients.

Modified Prosthetic Vascular Conduits

Atherosclerosis in the form of peripheral arterial disease results in significant morbidity. Surgical treatment options for peripheral arterial disease include angioplasty, endarterectomy, and bypass grafting. For bypass grafting, vein remains the conduit of choice; however, poor quality and limited availability have led to the use of prosthetic materials. Unfortunately, because of a lack of endothelium and compliance mismatch, neointimal hyperplasia develops aggressively, resulting in high failure rates. To improve graft patency, investigators have developed surgical, chemical, and biological graft modifications. This review describes common prosthetic materials, as well as approaches currently in use and under investigation to modify and improve prosthetic conduits for bypass grafting in an effort to improve graft patency rates.

Nitric oxide regulates the 26S proteasome in vascular smooth muscle cells

It is well established that nitric oxide (NO) inhibits vascular smooth muscle cell (VSMC) proliferation by modulating cell cycle proteins. The 26S proteasome is integral to protein degradation and tightly regulates cell cycle proteins. Therefore, we hypothesized that NO directly inhibits the activity of the 26S proteasome. The three enzymatic activities (chymotrypsin-like, trypsin-like and caspase-like) of the 26S proteasome were examined in VSMC. At baseline, caspase-like activity was approximately 3.5-fold greater than chymotrypsin- and trypsin-like activities. The NO donor S-nitroso-N-acetylpenicillamine (SNAP) significantly inhibited all three catalytically active sites in a time- and concentration-dependent manner (P<0.05). Caspase-like activity was inhibited to a greater degree (77.2% P<0.05). cGMP and cAMP analogs and inhibitors had no statistically significant effect on basal or NO-mediated inhibition of proteasome activity. Dithiothreitol, a reducing agent, prevented and reversed the NO-mediated inhibition of the 26S proteasome. Nitroso-cysteine analysis following S-nitrosoglutathione exposure revealed that the 20S catalytic core of the 26S proteasome contains 10 cysteines which were S-nitrosylated by NO. Evaluation of 26S proteasome subunit protein expression revealed differential regulation of the alpha and beta subunits in VSMC following exposure to NO. Finally, immunohistochemical analysis of subunit expression revealed distinct intracellular localization of the 26S proteasomal subunits at baseline and confirmed upregulation of distinct subunits following NO exposure. In conclusion, NO reversibly inhibits the catalytic activity of the 26S proteasome through S-nitrosylation and differentially regulates proteasomal subunit expression. This may be one mechanism by which NO exerts its effects on the cell cycle and inhibits cellular proliferation in the vasculature.

Isopropylamine NONOate (IPA/NO) moderates neointimal hyperplasia following vascular injury.

OBJECTIVE Isopropylamine NONOate (IPA/NO) is a nitroxyl (HNO) donor at physiologic pH. HNO is a positive inotrope and vasodilator, but little is known about its effect on neointimal hyperplasia. The aims of this study are to determine the effect of IPA/NO on endothelial and vascular smooth muscle cells (VSMC) in vitro and to determine if IPA/NO inhibits neointimal hyperplasia in vivo. METHODS VSMC were harvested from the abdominal aortas of male Sprague Dawley rats, and human umbilical vein endothelial cells were purchased from ATCC. In vitro, cellular proliferation was assessed by (3)H-thymidine incorporation, cell migration was assessed using the scrape assay, and cell death was assessed using Guava personal cell analysis (PCA). Cell cycle analysis was performed using propidium iodide staining and flow cytometry analysis. Protein expression was assessed using Western blot analysis. Phosphorylated proteins were assessed using immunoprecipitation and Western blot analysis. In vivo, the carotid artery injury model was performed on male Sprague Dawley rats treated with (n = 12) or without (n = 6) periadventitial IPA/NO (10 mg). Arteries harvested at 2 weeks were assessed for morphometrics using ImageJ. Inflammation was assessed using immunohistochemistry. Endothelialization was assessed by Evans blue staining of carotid arteries harvested 7 days after balloon injury from rats treated with (n = 6) or without (n = 3) periadventitial IPA/NO (10 mg). RESULTS In vitro, 1000 micromol/L IPA/NO inhibited both VSMC (38.7 +/- 4.5% inhibition vs control, P = .003) and endothelial cell proliferation (54.0 +/- 2.9% inhibition vs control, P < or = 0.001) without inducing cell death or inhibiting migration. In VSMC, this inhibition was associated with an S-phase cell cycle arrest and increased expression of cyclin A, cyclin D1, and the cyclin-dependent kinase inhibitor p21. No change was noted in the phosphorylation status of cdk2, cdk4, or cdk6 by IPA/NO. In rodents subjected to the carotid artery balloon injury model, IPA/NO caused significant reductions in neointimal area (298 +/- 20 vs 422 +/- 30, P < or = .001) and medial area (311 +/- 14 vs 449 +/- 16, P < or = .001) compared with injury alone, and reduced macrophage infiltration to 1.7 +/- 0.8 from 16.1 +/- 3.5 cells per high power field (P < or = .001). IPA/NO also prevented re-endothelialization compared with injury alone (55.9 +/- 0.5% nonendothelialized vs 21 +/- 4.4%, respectively, P = .001). Lastly, a 50% mortality rate was observed in the IPA/NO-treated groups. CONCLUSIONS In summary, while IPA/NO modestly inhibited neointimal hyperplasia by inhibiting VSMC proliferation and macrophage infiltration, it also inhibited endothelial cell proliferation and induced significant mortality in our animal model. Since HNO is being investigated as a treatment for congestive heart failure, our results raise some concerns about the use of IPA/NO in the vasculature and suggest that further studies be conducted on the safety of HNO donors in the cardiovascular system.

Heightened efficacy of nitric oxide-based therapies in type II diabetes mellitus and metabolic syndrome

Type II diabetes mellitus (DM) and metabolic syndrome are associated with accelerated restenosis following vascular interventions due to neointimal hyperplasia. The efficacy of nitric oxide (NO)-based therapies is unknown in these environments. Therefore, the aim of this study is to examine the efficacy of NO in preventing neointimal hyperplasia in animal models of type II DM and metabolic syndrome and examine possible mechanisms for differences in outcomes. Aortic vascular smooth muscle cells (VSMC) were harvested from rodent models of type II DM (Zucker diabetic fatty), metabolic syndrome (obese Zucker), and their genetic control (lean Zucker). Interestingly, NO inhibited proliferation and induced G0/G1 cell cycle arrest to the greatest extent in VSMC from rodent models of metabolic syndrome and type II DM compared with controls. This heightened efficacy was associated with increased expression of cyclin-dependent kinase inhibitor p21, but not p27. Using the rat carotid artery injury model to assess the efficacy of NO in vivo, we found that the NO donor PROLI/NO inhibited neointimal hyperplasia to the greatest extent in type II DM rodents, followed by metabolic syndrome, then controls. Increased neointimal hyperplasia correlated with increased reactive oxygen species (ROS) production, as demonstrated by dihydroethidium staining, and NO inhibited this increase most in metabolic syndrome and DM. In conclusion, NO was surprisingly a more effective inhibitor of neointimal hyperplasia following arterial injury in type II DM and metabolic syndrome vs. control. This heightened efficacy may be secondary to greater inhibition of VSMC proliferation through cell cycle arrest and regulation of ROS expression, in addition to other possible unidentified mechanisms that deserve further exploration.

Citric acid‐based elastomers provide a biocompatible interface for vascular grafts

Prosthetic vascular bypass grafting is associated with poor long-term patency rates. Herein, we report on the mid-term performance of expanded polytetrafluoroethylene (ePTFE) vascular grafts modified with a citric acid-based biodegradable elastomer. Through a spin-shearing method, ePTFE grafts were modified by mechanically coating a layer of poly(1,8 octanediol citrate) (POC) onto the luminal nodes and fibrils of the ePTFE. Control and POC-ePTFE grafts were implanted into the porcine carotid artery circulation as end-to-side bypass grafts. Grafts were assessed by duplex ultrasonography, magnetic resonance angiography, and digital subtraction contrast angiography and were all found to be patent with no hemodynamically significant stenoses. At 4 weeks, POC-ePTFE grafts were found to be biocompatible and resulted in a similar extent of neointimal hyperplasia as well as leukocyte and monocyte/macrophage infiltration as control ePTFE grafts. Furthermore, POC supported endothelial cell growth. Lastly, scanning electron microscopy confirmed the presence of POC on the ePTFE grafts at 4 weeks. Thus, these data reveal that surface modification of blood-contacting surfaces with POC results in a biocompatible surface that does not induce any untoward effects or inflammation in the vasculature. These findings are important as they will serve as the foundation for the development of a drug-eluting vascular graft.

YouTube is the Most Frequently Used Educational Video Source for Surgical Preparation.

OBJECTIVE The purpose of this study was to evaluate surgical preparation methods of medical students, residents, and faculty with special attention to video usage. DESIGN Following Institutional Review Board approval, anonymous surveys were distributed to participants. Information collected included demographics and surgical preparation methods, focusing on video usage. Participants were questioned regarding frequency and helpfulness of videos, video sources used, and preferred methods between videos, reading, and peer consultation. Statistical analysis was performed using SAS. SETTING Surveys were distributed to participants in the Department of Surgery at the University of Iowa Hospitals and Clinics, a tertiary care center in Iowa City, Iowa. PARTICIPANTS Survey participants included fourth-year medical students pursuing general surgery, general surgery residents, and faculty surgeons in the Department of Surgery. A total of 86 surveys were distributed, and 78 surveys were completed. This included 42 learners (33 residents, 9 fourth-year medical students) and 36 faculty. RESULTS The overall response rate was 91%; 90% of respondents reported using videos for surgical preparation (learners = 95%, faculty = 83%, p = NS). Regarding surgical preparation methods overall, most learners and faculty selected reading (90% versus 78%, p = NS), and fewer respondents reported preferring videos (64% versus 44%, p = NS). Faculty more often use peer consultation (31% versus 50%, p < 0.02). Among respondents who use videos (N = 70), the most used source was YouTube (86%). Learners and faculty use different video sources. Learners use YouTube and Surgical Council on Resident Education (SCORE) Portal more than faculty (YouTube: 95% versus 73%, p < 0.02; SCORE: 25% versus 7%, p < 0.05). Faculty more often use society web pages and commercial videos (society: 67% versus 38%, p < 0.03; commercial: 27% versus 5%, p < 0.02). CONCLUSIONS Most respondents reported using videos to prepare for surgery. YouTube was the preferred source. Posting surgical videos to YouTube may allow for maximal access to learners who are preparing for surgical cases.

A matched case-control study of IBD-associated colorectal cancer: IBD portends worse outcome

The effect of inflammatory bowel disease (IBD) on outcome in patients with colorectal cancer (CRC) remains unclear. Our objective is to evaluate oncologic outcomes of patients with IBD‐associated CRC.

Nitric oxide decreases activity and levels of the 11S proteasome activator PA28 in the vasculature

The 11S proteasome activator (PA28) binds to the 20S proteasome and increases its ability to degrade small peptides. Expression of PA28 subunits (α, β, γ) is induced by interferon-γ stimulation. Inflammation plays a role in the development of neointimal hyperplasia, and we have previously shown that nitric oxide (NO) reduces neointimal hyperplasia in animal models and 26S proteasome activity in rat aortic smooth muscle cells (RASMC). Here, we show that PA28 increased 26S proteasome activity in RASMC, as measured by a fluorogenic assay, and the NO donor S-nitroso N-acetylpenicillamine significantly inhibits this activation. This effect was abrogated by the reducing agents dithiothreitol and HgCl(2), suggesting that NO affects the activity of PA28 through S-nitrosylation. NO did not appear to affect PA28 levels or intracellular localization in RASMC in vitro. Three days following rat carotid artery balloon injury, levels of PA28α, β and γ subunits were decreased compared to uninjured control arteries (n=3/group) in vivo. The NO donor proline NONOate further decreased PA28α, β and γ levels by 1.9-, 2.3- and 3.4-fold, respectively, compared to uninjured control arteries. Fourteen days following arterial injury, levels of PA28α, β and γ subunits were increased throughout the arterial wall compared to uninjured control arteries, but were greatest for the α and β subunits. NO continued to decrease the levels of all three PA28 subunits throughout the arterial wall at this time point. Since the PA28 subunits are involved in the breakdown of peptides during inflammation, PA28 inhibition may be one mechanism by which NO inhibits neointimal hyperplasia.

Volvulus of the Small Bowel and Colon

Abstract Volvulus of the intestines may involve either the small bowel or colon. In the pediatric population, small bowel volvulus is more common, while in the adult population, colonic volvulus is more often seen. The two most common types of colonic volvulus include sigmoid and cecal volvulus. Prompt diagnosis and treatment is imperative, otherwise bowel ischemia may ensue. Treatment often involves emergent surgical exploration and bowel resection.