Pasithorn A. Suwanabol

The Novel Function of Advanced Glycation End Products in Regulation of MMP-9 Production

BACKGROUND Advanced glycation end products (AGEs), formed from proteins and peptides by nonenzymatic glycoxidation after contact with aldose sugars, have been implicated in the pathogenesis of age-related cardiac and vascular dysfunction. Our previous study demonstrated significantly elevated levels of AGE and the receptor for AGE (RAGE) in human abdominal aortic aneurysm (AAA) tissues. Inhibition of AGE signaling by targeted gene deletion of RAGE markedly reduced the development of aneurysm in a mouse model of AAA. We also showed that AGE may stimulate aneurysm formation by promoting metalloproteinase (MMP)-9 expression. In this study, we investigated the molecular mechanism underlying this novel function of AGE. METHODS The murine macrophage cell line RAW 264.7 was pretreated with AGE, TGF-β, and MAPK inhibitors. The protein was collected for Western blot analysis. Culture supernatants were collected to determine MMP-9 activity by gelatin zymography. RESULTS We found that AGE induced the production of MMP-9 in macrophages in a dose-dependent manner. This induction of MMP-9 was markedly diminished by pretreatment with TGF-β. To delineate the underlying molecular mechanism, we showed that AGE increased phosphorylation of p44/42 ERK, p38, JNK, and PI3K in macrophages. Moreover, AGE induced active p65 subunit of NF- κB. Inhibition of ERK (UO126) or p38 (SB203580), but not PI3K (LY294002 or wortmannin), blocked AGE-induced MMP-9 expression. In contrast, inhibition of JNK (SP-600125) significantly enhanced the stimulatory effect of AGE on MMP-9. Furthermore, TGF-β suppressed AGE-induced expression of the active p65 subunit of NF-κB. CONCLUSIONS Our data indicate that AGE induces MMP-9 through activation of ERK, p38 mitogen-activated protein and NF-κB, a pathway that is antagonized by TGF-β. This finding in conjunction with previously reported AGE functions in inflammation suggests that anti-AGE therapies could be effective in the prevention of human AAA development and progression.

Local drug delivery to prevent restenosis

INTRODUCTION Despite significant advances in vascular biology, bioengineering, and pharmacology, restenosis remains a limitation to the overall efficacy of vascular reconstructions, both percutaneous and open. Although the pathophysiology of intimal hyperplasia is complex, a number of drugs and molecular tools have been identified that can prevent restenosis. Moreover, the focal nature of this process lends itself to treatment with local drug administration. This article provides a broad overview of current and future techniques for local drug delivery that have been developed to prevent restenosis after vascular interventions. METHODS A systematic electronic literature search using PubMed was performed for all accessible published articles through September 2012. In an effort to remain current, additional searches were performed for abstracts presented at relevant societal meetings, filed patents, clinical trials, and funded National Institutes of Health awards. RESULTS The efficacy of local drug delivery has been demonstrated in the coronary circulation with the current clinical use of drug-eluting stents. Until recently, however, drug-eluting stents were not found to be efficacious in the peripheral circulation. Further pursuit of intraluminal devices has led to the development of balloon-based technologies, with a recent surge in trials involving drug-eluting balloons. Early data appear encouraging, particularly for treatment of superficial femoral artery lesions, and several devices have recently received the Conformité Européene mark in Europe. Investigators have also explored the periadventitial application of biomaterials containing antirestenotic drugs, an approach that could be particularly useful for surgical bypass or endarterectomy. In the past, systemic drug delivery has been unsuccessful; however, there has been recent exploration of intravenous delivery of drugs designed specifically to target injured or reconstructed arteries. Our review revealed a multitude of additional interesting strategies, including >65 new patents issued during the past 2 years for approaches to local drug delivery focused on preventing restenosis. CONCLUSIONS Restenosis after intraluminal or open vascular reconstruction remains an important clinical problem. Success in the coronary circulation has not translated into solutions for the peripheral arteries. However, our literature review reveals a number of promising approaches, including drug-eluting balloons, periadventitial drug delivery, and targeted systemic therapies. These and other innovations suggest that the future is bright and that a solution for preventing restenosis in peripheral vessels will soon be at hand.

TGF-β and Restenosis Revisited: A Smad Link

Despite novel surgical therapies for the treatment of atherosclerosis, restenosis continues to be a significant impediment to the long-term success of vascular interventions. Transforming growth factor-beta (TGF-β), a family of cytokines found to be up-regulated at sites of arterial injury, has long been implicated in restenosis; a role that has largely been attributed to TGF-β-mediated vascular fibrosis. However, emerging data indicate that the role of TGF-β in intimal thickening and arterial remodeling, the critical components of restenosis, is complex and multidirectional. Recent advancements have clarified the basic signaling pathway of TGF-β, making evident the need to redefine the precise role of this family of cytokines and its primary signaling pathway, Smad, in restenosis. Unraveling TGF-β signaling in intimal thickening and arterial remodeling will pave the way for a clearer understanding of restenosis and the development of innovative pharmacological therapies.

Syndromes Associated With the Deep Veins: Phlegmasia Cerulea Dolens, May-Thurner Syndrome, and Nutcracker Syndrome

Although phlegmasia cerulea dolens, May-Thurner syndrome, and nutcracker syndrome are rare entities, knowledge of these syndromes associated with the deep veins is essential. This study presents current management of these disorders, including diagnostic and interventional strategies. Endovascular techniques have evolved and now play a significant role in the treatment of both phlegmasia cerulea dolens and May-Thurner syndrome. However, endovascular therapy for nutcracker syndrome remains untested.

Transforming growth factor-β increases vascular smooth muscle cell proliferation through the Smad3 and extracellular signal-regulated kinase mitogen-activated protein kinases pathways

INTRODUCTION We have previously demonstrated that transforming growth factor-β (TGF-β) in the presence of elevated levels of Smad3, its primary signaling protein, stimulates rat vascular smooth muscle cell (VSMC) proliferation and intimal hyperplasia. The mechanism is partly through the nuclear exportation of phosphorylated cyclin-dependent kinase inhibitor p27. The objective of this study is to clarify the downstream pathways through which Smad3 produces its proliferative effect. Specifically, we evaluated the role of extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) in TGF-β-induced VSMC proliferation. METHODS Cultured rat aortic VSMCs were incubated with TGF-β at varying concentrations and times, and phosphorylated ERK was measured by Western blotting. Smad3 was enhanced in VSMCs using an adenovirus expressing Smad3 or inhibited with small interfering RNA (siRNA). For in vivo experiments, male Sprague-Dawley rats underwent carotid balloon injury, followed by intraluminal infection with an adenovirus expressing Smad3. Arteries were harvested at 3 days and subjected to immunohistochemistry for Smad3, phospho-ERK MAPK, and proliferating cell nuclear antigen. RESULTS In cultured VSMCs, TGF-β induced activation and phosphorylation of ERK MAPK in a time-dependent and concentration-dependent manner. Overexpression of the signaling protein Smad3 enhanced TGF-β-induced activation of ERK MAPK, whereas inhibition of Smad3 with a siRNA blocked ERK MAPK phosphorylation in response to TGF-β. These data suggest that Smad3 acts as a signaling intermediate between TGF-β and ERK MAPK. Inhibition of ERK MAPK activation with PD98059 completely blocked the ability of TGF-β/Smad3 to stimulate VSMC proliferation, demonstrating the importance of ERK MAPK in this pathway. Immunoprecipitation of phospho-ERK MAPK and blotting with Smad3 revealed a physical association, suggesting that activation of ERK MAPK by Smad3 requires a direct interaction. In an in vivo rat carotid injury model, overexpression of Smad3 resulted in an increase in phosphorylated ERK MAPK as well as increased VSMC proliferation as measured by proliferating cell nuclear antigen. CONCLUSIONS Our findings demonstrate a mechanism through which TGF-β stimulates VSMC proliferation. Although TGF-β has been traditionally identified as an inhibitor of proliferation, our data suggest that TGF-β enhances VSMC proliferation through a Smad3/ERK MAPK signaling pathway. These findings at least partly explain the mechanism by which TGF-β enhances intimal hyperplasia. Knowledge of this pathway provides potential novel targets that may be used to prevent restenosis.

Basic research studyFrom the Midwestern Vascular Surgical SocietyTransforming growth factor-β increases vascular smooth muscle cell proliferation through the Smad3 and extracellular signal-regulated kinase mitogen-activated protein kinases pathways

INTRODUCTION We have previously demonstrated that transforming growth factor-β (TGF-β) in the presence of elevated levels of Smad3, its primary signaling protein, stimulates rat vascular smooth muscle cell (VSMC) proliferation and intimal hyperplasia. The mechanism is partly through the nuclear exportation of phosphorylated cyclin-dependent kinase inhibitor p27. The objective of this study is to clarify the downstream pathways through which Smad3 produces its proliferative effect. Specifically, we evaluated the role of extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) in TGF-β-induced VSMC proliferation. METHODS Cultured rat aortic VSMCs were incubated with TGF-β at varying concentrations and times, and phosphorylated ERK was measured by Western blotting. Smad3 was enhanced in VSMCs using an adenovirus expressing Smad3 or inhibited with small interfering RNA (siRNA). For in vivo experiments, male Sprague-Dawley rats underwent carotid balloon injury, followed by intraluminal infection with an adenovirus expressing Smad3. Arteries were harvested at 3 days and subjected to immunohistochemistry for Smad3, phospho-ERK MAPK, and proliferating cell nuclear antigen. RESULTS In cultured VSMCs, TGF-β induced activation and phosphorylation of ERK MAPK in a time-dependent and concentration-dependent manner. Overexpression of the signaling protein Smad3 enhanced TGF-β-induced activation of ERK MAPK, whereas inhibition of Smad3 with a siRNA blocked ERK MAPK phosphorylation in response to TGF-β. These data suggest that Smad3 acts as a signaling intermediate between TGF-β and ERK MAPK. Inhibition of ERK MAPK activation with PD98059 completely blocked the ability of TGF-β/Smad3 to stimulate VSMC proliferation, demonstrating the importance of ERK MAPK in this pathway. Immunoprecipitation of phospho-ERK MAPK and blotting with Smad3 revealed a physical association, suggesting that activation of ERK MAPK by Smad3 requires a direct interaction. In an in vivo rat carotid injury model, overexpression of Smad3 resulted in an increase in phosphorylated ERK MAPK as well as increased VSMC proliferation as measured by proliferating cell nuclear antigen. CONCLUSIONS Our findings demonstrate a mechanism through which TGF-β stimulates VSMC proliferation. Although TGF-β has been traditionally identified as an inhibitor of proliferation, our data suggest that TGF-β enhances VSMC proliferation through a Smad3/ERK MAPK signaling pathway. These findings at least partly explain the mechanism by which TGF-β enhances intimal hyperplasia. Knowledge of this pathway provides potential novel targets that may be used to prevent restenosis.

TGF-β and Smad3 modulate PI3K/Akt signaling pathway in vascular smooth muscle cells

Transforming growth factor-β (TGF-β) is upregulated at the time of arterial injury; however, the mechanism through which TGF-β enhances the development of intimal hyperplasia is not clear. Recent studies from our laboratory suggest that in the presence of elevated levels of Smad3, TGF-β stimulates smooth muscle cell (SMC) proliferation. This is a novel phenomenon in that TGF-β has traditionally been known as a potent inhibitor of cellular proliferation. In these studies we explore the signaling pathways through which TGF-β mediates its proliferative effect in vascular SMCs. We found that TGF-β phosphorylates and activates Akt in a time-dependent manner, and this effect is significantly enhanced by overexpression of Smad3. Furthermore, both chemical and molecular inhibition of Smad3 can reverse the effect of TGF-β on Akt. Although we found numerous signaling pathways that might function as intermediates between Smad3 and Akt, p38 appeared the most promising. Overexpression of Smad3 enhanced p38 phosphorylation and inhibition of p38 with a chemical inhibitor or a small interfering RNA blocked TGF-β-induced Akt phosphorylation. Moreover, TGF-β/Smad3 enhancement of SMC proliferation was blocked by inhibition of p38. Phosphorylation of Akt by TGF-β/Smad3 was not dependent on gene expression or protein synthesis, and immunoprecipitation studies revealed a physical association among p38, Akt, and Smad3 suggesting that activation requires a direct protein-protein interaction. Our findings were confirmed in vivo where overexpression of Smad3 in a rat carotid injury model led to enhancement of p-p38, p-Akt, as well as SMC proliferation. Furthermore, inhibition of p38 in vivo led to decreased Akt phosphorylation and SMC proliferation. In summary, our studies reveal a novel pathway whereby TGF-β/Smad3 stimulates SMC proliferation through p38 and Akt. These findings provide a potential mechanism for the substantial effect of TGF-β on intimal hyperplasia and suggest new targets for chemical or molecular prevention of vascular restenosis.

TGF-β/Smad3 inhibit vascular smooth muscle cell apoptosis through an autocrine signaling mechanism involving VEGF-A

We have previously shown that in the presence of elevated Smad3, transforming growth factor-β (TGF-β) transforms from an inhibitor to a stimulant of vascular smooth muscle cell (SMC) proliferation and intimal hyperplasia (IH). Here we identify a novel mechanism through which TGF-β/Smad3 also exacerbates IH by inhibiting SMC apoptosis. We found that TGF-β treatment led to inhibition of apoptosis in rat SMCs following viral expression of Smad3. Conditioned media from these cells when applied to naive SMCs recapitulated this effect, suggesting an autocrine pathway through a secreted factor. Gene array of TGF-β/Smad3-treated cells revealed enhanced expression of vascular endothelial growth factor (VEGF), a known inhibitor of endothelial cell apoptosis. We then evaluated whether VEGF is the secreted mediator responsible for TGF-β/Smad3 inhibition of SMC apoptosis. In TGF-β/Smad3-treated cells, VEGF mRNA and protein as well as VEGF secretion were increased. Moreover, recombinant VEGF-A inhibited SMC apoptosis and a VEGF-A-neutralizing antibody reversed the inhibitory effect of conditioned media on SMC apoptosis. Stimulation of SMCs with TGF-β led to the formation of a complex of Smad3 and hypoxia-inducible factor-1α (HIF-1α) that in turn activated the VEGF-A promoter and transcription. In rat carotid arteries following arterial injury, Smad3 and VEGF-A expression were upregulated. Moreover, Smad3 gene transfer further enhanced VEGF expression as well as inhibited SMC apoptosis. Finally, blocking either the VEGF receptor or Smad3 signaling in injured carotid arteries abrogated the inhibitory effect of Smad3 on vascular SMC apoptosis. Taken together, our study reveals that following angioplasty, elevation of both TGF-β and Smad3 leads to SMC secretion of VEGF-A that functions as an autocrine inhibitor of SMC apoptosis. This novel pathway provides further insights into the role of TGF-β in the development of IH.

LATE MANIFESTATION OF TRACHEAL RUPTURE AFTER THYROIDECTOMY: CASE REPORT AND LITERATURE REVIEW

OBJECTIVE To report an extremely rare case of delayed tracheal rupture after thyroidectomy and to review the existing related literature. METHODS We present the history, clinical findings, radiographic evaluation, management, and intraoperative findings in a patient who presented with subcutaneous emphysema 9 days after total thyroidectomy. In addition, we review the literature and discuss the diagnostic challenges as well as management options. RESULTS A 17-year-old female patient underwent a total thyroidectomy for Graves disease. On postoperative day 9, the patient presented with face and neck swelling attributable to subcutaneous emphysema. After conservative management failed, the patient underwent surgical exploration of the neck, which revealed a 2.5-cm linear vertical tear in the anterior aspect of the trachea, with no evidence of necrosis. The tear had viable edges and was primarily repaired with use of muscle flap reinforcement. The patient recovered with no other complications. CONCLUSION Delayed tracheal rupture should be suspected in all patients who present with subcutaneous emphysema after a thyroid surgical procedure. Review of the pertinent literature suggests that conservative management is suitable in patients with a stable condition. Surgical repair is indicated in those patients who fail to demonstrate clinical improvement.

Clinical Implications of Non–Contrast-Enhanced Computed Tomography for Follow-Up After Endovascular Abdominal Aortic Aneurysm Repair

BACKGROUND There is growing concern over the long-term radiation exposure from serial computed tomographic (CT) scan follow-up after endovascular aneurysm repair (EVAR) of abdominal aortic aneurysms (AAAs). Screening for endoleaks with non-contrast-enhanced volumetric CT has been shown to significantly reduce radiation doses. We evaluated the use of NCT as the primary method of follow-up after EVAR of AAAs. METHODS Our institutional post-EVAR CT protocol consisted of contrast-enhanced CT angiography (CTA) 1 month after repair, followed by NCT at 3 or 6 and 12 months, and annually thereafter. At each follow-up scan, immediate 3-dimensional volume analysis was performed. If the volume change was <2%, NCT follow-up was continued. If the volume increased by ≥2% on nonenhanced images, contrast-enhanced CT was performed immediately to identify potential endoleaks. All images were reviewed by an experienced cardiovascular radiologist. End points included identification of endoleak, reintervention, and rupture. RESULTS Over a 7-year period, 126 patients were followed. Serial CTA was performed in 59 patients, while 67 patients were followed with the NCT protocol. The mean follow-up was 2.07 years. There were no differences in age, sex, or initial aneurysm volume or size. There were 35 total endoleaks identified. Twenty of these were early endoleaks (<30 days post-EVAR). The remaining 15 leaks were late in nature (10 in the contrast group and 5 in the noncontrast group; P=0.17). NCT aneurysm sac volume changes prompted contrasted studies in all 5 late leaks. The mean volume change was 11.2 cm3, an average change of 5.88%. These findings were not significantly different than the late leaks found by routine contrast studies (8.9 cm3; 4.98% [P=0.58]). There were no delayed ruptures or emergent reinterventions in the NCT group. CONCLUSIONS Serial NCT appears to be safe and effective as the sole means of follow-up after EVAR for AAAs. AAA volume increases of ≥2% should prompt further contrast-enhanced CT imaging. Changes of <2% can be safely followed with serial NCT. This protocol requires dedicated cardiovascular radiologist involvement, and patients should be retained in the radiology suite until real-time image evaluation can be completed.