Daniel A. Popowich

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.

HIV and anal cancer outcomes: A single institution's experience

PURPOSE: The purpose of this study is to identify the effect of HIV status on outcome of treatment for squamous-cell carcinoma of the anal canal. METHODS: A retrospective review was performed on all patients with squamous-cell carcinoma of the anal canal treated at a single academic institution between January 1996 and December 2006. RESULTS: Our search identified 87 (21 HIV-positive) patients who had invasive squamous-cell cancer. The median follow-up was 38 months. Eighty-five percent of HIV-negative patients and 81 percent of HIV-positive were identified as complete responders at 6 weeks after completion of combined modality therapy. Eight percent of HIV-negative and 29 percent of HIV-positive patients developed recurrent disease after 6 months (P = 0.0009). Overall survival for HIV-negative and HIV-positive patients was 71 percent and 73 percent, respectively. CONCLUSIONS: HIV-positive patients respond equally to combined modality therapy but have recurrences more frequently than patients who are HIV negative. Overall survival in these two groups is equivalent.

Regulation of reactive oxygen species by p53: implications for nitric oxide-mediated apoptosis

Nitric oxide (NO) induces vascular smooth muscle cell (VSMC) apoptosis in part through activation of p53. Traditionally, p53 has been thought of as the gatekeeper, determining if a cell should undergo arrest and repair or apoptosis following exposure to DNA-damaging agents, depending on the severity of the damage. However, our laboratory previously demonstrated that NO induces apoptosis to a much greater extent in p53(-/-) compared with p53(+/+) VSMC. Increased reactive oxygen species (ROS) within VSMC has been shown to induce VSMC apoptosis, and recently it was found that the absence of, or lack of, functional p53 leads to increased ROS and oxidative stress within different cell types. This study investigated the differences in intracellular ROS levels between p53(-/-) and p53(+/+) VSMC and examined if these differences were responsible for the increased susceptibility to NO-induced apoptosis observed in p53(-/-) VSMC. We found that p53 actually protects VSMC from NO-induced apoptosis by increasing antioxidant protein expression [i.e., peroxiredoxin-3 (PRx-3)], thereby reducing ROS levels and cellular oxidative stress. We also observed that the NO-induced apoptosis in p53(-/-) VSMC was largely abrogated by pretreatment with catalase. Furthermore, when the antioxidant protein PRx-3 and its specific electron acceptor thioredoxin-2 were silenced within p53(+/+) VSMC with small-interfering RNA, not only did these cells exhibit greater ROS production, but they also exhibited increased NO-induced apoptosis similar to that observed in p53(-/-) VSMC. These findings suggest that ROS mediate NO-induced VSMC apoptosis and that p53 protects VSMC from NO-induced apoptosis by decreasing intracellular ROS. This research demonstrates that p53 has antioxidant functions in stressed cells and also suggests that p53 has antiapoptotic properties.

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.

Effect of Nitric Oxide on Neointimal Hyperplasia based on Sex and Hormone Status

Nitric oxide (NO)-based therapies decrease neointimal hyperplasia; however, studies have been performed only in male animal models. Thus, we sought to evaluate the effect of NO on vascular smooth muscle cells (VSMC) in vitro and neointimal hyperplasia in vivo based on sex and hormone status. In hormone-replete medium, male VSMC proliferated at greater rates than female VSMC. In hormone-depleted medium, female VSMC proliferated at greater rates than male VSMC. However, in both hormone environments, NO inhibited proliferation and migration to a greater extent in male compared to female VSMC. These findings correlated with greater G₀/G₁ cell cycle arrest and changes in cell cycle protein expression in male compared to female VSMC after exposure to NO. Next, the rat carotid artery injury model was used to assess the effect of NO on neointimal hyperplasia in vivo. Consistent with the in vitro data, NO was significantly more effective at inhibiting neointimal hyperplasia in hormonally intact males compared to females using weight-based dosing. An increased weight-based dose of NO in females was able to achieve efficacy equal to that in males. Surprisingly, NO was less effective at inhibiting neointimal hyperplasia in castrated animals of both sexes. In conclusion, these data suggest that NO inhibits neointimal hyperplasia more effectively in males compared to females and in hormonally intact compared to castrated rats, indicating that the effects of NO in the vasculature may be sex- and hormone-dependent.

Insulin enhances the effect of nitric oxide at inhibiting neointimal hyperplasia in a rat model of type 1 diabetes

Diabetes confers greater restenosis from neointimal hyperplasia following vascular interventions. While localized administration of nitric oxide (NO) is known to inhibit neointimal hyperplasia, the effect of NO in type 1 diabetes is unknown. Thus the aim of this study was to determine the efficacy of NO following arterial injury, with and without exogenous insulin administration. Vascular smooth muscle cells (VSMC) from lean Zucker (LZ) rats were exposed to the NO donor, DETA/NO, following treatment with different glucose and/or insulin concentrations. DETA/NO inhibited VSMC proliferation in a concentration-dependent manner to a greater extent in VSMC exposed to normal-glucose vs. high-glucose environments, and even more effectively in normal-glucose/high-insulin and high-glucose/high-insulin environments. G(0)/G(1) cell cycle arrest and cell death were not responsible for the enhanced efficacy of NO in these environments. Next, type 1 diabetes was induced in LZ rats with streptozotocin. The rat carotid artery injury model was performed. Type 1 diabetic rats experienced no significant reduction in neointimal hyperplasia following arterial injury and treatment with the NO donor PROLI/NO. However, daily administration of insulin to type 1 diabetic rats restored the efficacy of NO at inhibiting neointimal hyperplasia (60% reduction, P < 0.05). In conclusion, these data demonstrate that NO is ineffective at inhibiting neointimal hyperplasia in an uncontrolled rat model of type 1 diabetes; however, insulin administration restores the efficacy of NO at inhibiting neointimal hyperplasia. Thus insulin may play a role in regulating the downstream beneficial effects of NO in the vasculature.

Nitric Oxide: What a Vascular Surgeon Needs to Know

Atherosclerosis in the form of peripheral arterial disease results in significant morbidity and mortality. Surgical treatment options for peripheral arterial disease include angioplasty with and without stenting, endarterectomy, and bypass grafting. Unfortunately, all of these procedures injure the vascular endothelium, which impairs its ability to produce nitric oxide (NO) and ultimately leads to neointimal hyperplasia and restenosis. To improve on current patency rates after vascular procedures, investigators are engaged in research to improve the bioavailability of NO at the site of vascular injury in an attempt to reduce the risk of thrombosis and restenosis after successful revascularization. This article reviews some of the previous research that has aimed to improve NO bioavailability after vascular procedures whether through systemic or local delivery, as well as to describe some of the NO-releasing products that are currently undergoing study for use in clinical practice.

Fistula-associated Anorectal Cancer in the Setting of Crohn’s Disease

BACKGROUND: Cancer arising from perianal fistulas in patients with Crohn’s disease is rare. There are only a small series of articles that describe sporadic cases of perianal cancer in Crohn’s disease fistulas. Therefore, there are no clear guidelines on how to appropriately screen patients at risk and choose proper management. OBJECTIVE: The purpose of this study was to describe patients diagnosed with cancer in perianal fistulas in the setting of Crohn’s disease. DESIGN: The study involved an institutional review board–approved retrospective review of medical charts of patients with perianal Crohn’s disease. The data extracted from patient charts included demographic and clinical characteristics. SETTINGS: Patients seen at the Mount Sinai Medical Center were included. PATIENTS: We identified patients who were diagnosed with perianal cancer in biopsies of fistula tracts. MAIN OUTCOME MEASURES: We observed the number of patients with Crohns disease who had fistulas, cancer in fistula tract, and time to diagnosis. RESULTS: The charts of 2382 patients with fistulizing perianal Crohn’s disease were reviewed. Cancer in a fistula tract was diagnosed in 19 (0.79%) of these patients, 9 with squamous-cell carcinoma and 10 with adenocarcinoma. The majority of the 19 patients (68%) had symptoms typical of perianal fistula. The mean time from diagnosis of Crohn’s disease to fistula diagnosis and from fistula diagnosis to cancer diagnosis was 19.4 and 6.0 years. In 5 patients (26%), cancer was not diagnosed in the first biopsy obtained from the fistula tract. LIMITATIONS: This is a retrospective chart review of a rare outcome; the results may not be generalizable. CONCLUSIONS: Routine biopsies of long-standing fistula tracts in patients with Crohn’s disease should be strongly considered and may yield an earlier diagnosis of cancer in the fistula tracts.

Multimodality therapy in unresectable colorectal cancer

Mathis et al. aimed to determine the effect of multimodality therapy on recurrence and survival in patients with locally advanced colorectal cancer. Use of multimodality treatment led to excellent local control with five year disease-free and overall survival rates comparable to that of stage-matched resectable colorectal cancers.