Improving arteriovenous fistula patency: Transdermal delivery of diclofenac reduces cannulation-dependent neointimal hyperplasia via AMPK activation.

Creation of an autologous arteriovenous fistula (AVF) for vascular access in haemodialysis is the modality of choice. However neointimal hyperplasia and loss of the luminal compartment result in AVF patency rates of ~60% at 12months. The exact cause of neointimal hyperplasia in the AVF is poorly understood. Vascular trauma has long been associated with hyperplasia. With this in mind in our rabbit model of AVF we simulated cannulation autologous to that undertaken in vascular access procedures and observed significant neointimal hyperplasia as a direct consequence of cannulation. The neointimal hyperplasia was completely inhibited by topical transdermal delivery of the non-steroidal anti-inflammatory (NSAID) diclofenac. In addition to the well documented anti-inflammatory properties we have identified novel anti-proliferative mechanisms demonstrating diclofenac increases AMPK-dependent signalling and reduced expression of the cell cycle protein cyclin D1. In summary prophylactic transdermal delivery of diclofenac to the sight of AVF cannulation prevents adverse neointimal hyperplasic remodelling and potentially offers a novel treatment option that may help prolong AVF patency and flow rates.

Inhibition of cyclooxygenase-2 modulates phenotypic switching of vascular smooth muscle cells during increased aortic blood flow.

This study investigates the interactions between cyclooxygenase (COX-2) and vascular smooth muscle cell (VSMC) phenotypic switching, the two important coupling mechanisms of the vasculature on arterial remodeling in response to high laminal shear stress. High aortic blood flow was induced by creating a fistula in the abdominal aorta and the adjacent IVC of anesthetized rats. Celecoxib, a selective COX-2 inhibitor (25 mg/kg/day), was fed in the chow, and animals were killed 8 weeks later. Blood flow, vasoreactivity and morphological changes in the aorta proximal to the fistula were measured. Concentrations of collagen, expression of desmin and smooth muscle myosin heavy chain (SM-MHC)-II in the aorta were determined. Celecoxib significantly increased aortic blood flow and reduced the contraction responses of aorta. Decreased medial thickness, presence of intimal thickening and derangement of elastic lamina were found in the aortic section of celecoxib-treated animals. Celecoxib significantly reduced the tissue content of collagen and upregulated expression of SM-MHC-II and desmin in the high-flow aorta. Inhibition of COX-2 enzymatic activity in the aorta exposed to higher blood flow resulted in increased blood flow and vascular remodeling. These functional changes were accomplished by VSMC phenotypic switching and reduced biosynthesis of collagen.

Macrophage migration inhibitory factor reduces apoptosis in cerebral arteriovenous malformations.

PURPOSE: To investigate the expression of macrophage migration inhibitory factor (MIF) in human brain arteriovenous malformations (AVM). MATERIALS AND METHODS: Twelve AVM specimens were obtained from patients who did not received preoperative embolization. MIF levels were measured by Western blot and matrix metalloproteinase 9 (MMP9) levels were measured by reverse transcription PCR. The expression of MIF in brain AVMs was also evaluated by immunohistochemistry and was correlated with apoptosis and the expression of cleaved caspase-3 and MMP9. RESULTS: The expression of MIF, MMP9, and cleaved caspase-3 was elevated in brain AVM vessels. High levels of MIF were primarily found in the endothelium and adventitia, whereas apoptotic cells were concentrated in the smooth muscle layer. CONCLUSIONS: Abnormal apoptosis may be involved in the pathogenesis of brain AVM. In addition, increased MIF expression could play an important role regulating the homeostasis of AVM vessels.

Regional and systemic hemodynamic responses following the creation of a murine arteriovenous fistula.

The study of hemodynamic alterations following the creation of an arteriovenous fistula (AVF) is relevant to vascular adaptive responses and hemodialysis access dysfunction. This study examined such alterations in a murine AVF created by anastomosing the carotid artery to the jugular vein. AVF blood flow was markedly increased due to reduced AVF vascular resistance. Despite such markedly increased basal blood flow, AVF blood flow further increased in response to acetylcholine. This AVF model exhibited increased cardiac output and decreased systemic vascular resistance; the kidney, in contrast, exhibited decreased blood flow and increased vascular resistance. Augmentation in AVF blood flow was attended by increased arterial heme oxygenase-1 (HO-1) mRNA and protein expression, the latter localized to smooth muscle cells of the AVF artery; AVF blood flow was substantially reduced in HO-1(-/-) mice compared with HO-1(+/+) mice. Finally, in a murine model of a representative disease known to exhibit impaired hemodynamic responses (sickle cell disease), the creation of an AVF was attended by decreased AVF flow and impaired AVF function. We conclude that this AVF model exhibits markedly increased AVF blood flow, a vasodilatory reserve capacity, increased cardiac output, decreased renal blood flow, and a dependency on intact hemodynamic responses, in general, and HO-1 expression, in particular, in achieving and maintaining AVF blood flow. We suggest that these findings support the utility of this model in investigating the basis for and the consequences of hemodynamic stress, including shear stress, and the pathobiology of hemodialysis AVF dysfunction.

Molecular imaging of matrix metalloproteinase activation to predict murine aneurysm expansion in vivo.

UNLABELLED: Rupture and dissection are major causes of morbidity and mortality in arterial aneurysm and occur more frequently in rapidly expanding aneurysms. Current imaging modalities provide little information on aneurysm beyond size. Matrix metalloproteinase (MMP) activation plays a key role in the pathogenesis of aneurysm. We investigated whether imaging MMP activation in aneurysm helps predict its propensity to expansion. METHODS: We used a model of carotid aneurysm in apolipoprotein E-deficient (apoE(-/-)) mice. Radiotracers with specificity for activated MMPs were used to detect and quantify MMP activation by micro-SPECT/CT in vivo. Tracer uptake was confirmed by autoradiography and gamma-well counting, and specificity was demonstrated using an excess of unlabeled precursor and a specific MMP inhibitor. RESULTS: We demonstrated that several MMPs are expressed with distinct temporal patterns in aneurysm. Significant focal uptake was observed in aneurysmal carotid arteries, peaking at 4 wk after aneurysm induction. In a group of animals imaged serially at 2 and 4 wk after aneurysm induction, MMP tracer uptake at 2 wk correlated well with the vessel area assessed by histology at 4 wk. CONCLUSION: Molecular imaging of MMP activation is a useful experimental, and potentially clinical, tool to noninvasively predict the propensity of an aneurysm to expansion in vivo.

Systemic expression of matrix metalloproteinase-9 in patients with cerebral arteriovenous malformations.

OBJECTIVE: Increased expression angiogenic factors, such as matrix metalloproteinases (MMPs), are associated with the formation of cerebral arteriovenous malformations (AVMs). The objective of this study was to determine plasma levels of MMP-9 of patients with AVMs. METHODS: Blood samples were drawn from 15 patients with AVMs before treatment, 24 hours postembolization, 24 hours postresection, and 30 days postresection. Blood samples were also obtained from 30 healthy controls. Plasma MMP-9 concentrations were measured via enzyme-linked immunosorbent assay. RESULTS: The mean plasma MMP-9 level in AVM patients at baseline was significantly higher than in control patients: 108.04 +/- 16.11 versus 41.44 +/- 2.44 ng/mL, respectively. The mean plasma MMP-9 level 1 day after embolization increased to 172.35 +/- 53.76 ng/mL, which was not significantly elevated over pretreatment levels. One day after resection, plasma MMP-9 levels increased significantly over pretreatment levels to 230.97 +/- 51.00 ng/mL. Mean plasma MMP-9 concentrations 30 days after resection decreased to 92.8 +/- 18.7 ng/mL, which was not different from pretreatment levels but was still significantly elevated over control levels. MMP-9 levels did not correlate with patient sex, age, presentation, or AVM size. CONCLUSION: Plasma MMP-9 levels are significantly elevated over controls at baseline, increase significantly immediately after surgery, and decrease to pretreatment levels during follow-up.

Flow loading induces macrophage antioxidative gene expression in experimental aneurysms.

OBJECTIVE: Reactive oxygen species may act as proinflammatory mediators in abdominal aortic aneurysm (AAA) disease. Flow loading increases antioxidative enzyme expression and limits reactive oxygen species production in vascular smooth muscle cells in vitro, limits experimental AAA enlargement in rodent models, and is indirectly associated with reduced clinical AAA risk. We attempted to determine the mechanism or mechanisms by which flow loading limits AAA enlargement. METHODS AND RESULTS: Rodent AAAs were flow loaded via femoral arteriovenous fistula creation. Aortic wall shear stress and relative wall strain were significantly higher in flow-loaded rodents. Flow loading reduced AAA diameter by 26% despite evidence of flow-mediated aortic enlargement proximal to the aneurysmal segment. Messenger RNA from AAA tissue was harvested for cDNA labeling and hybridization to a 384-clone DNA microarray. Twenty-nine genes were differentially expressed (relative intensity/relative intensity of control ratio >1.5 and <0.67) in flow-loaded compared with normal flow AAA tissue, including heme oxygenase 1 (HO-1). Increased HO-1 expression was confirmed via reverse transcriptase-polymerase chain reaction. Immunohistochemistry localized HO-1 expression to infiltrative macrophages. alpha-Tocopherol was found to be as effective as flow loading in limiting AAA enlargement. Flow loading and alpha-tocopherol therapy reduced AAA reactive oxygen species production. CONCLUSIONS: Flow loading may attenuate AAA enlargement via wall shear or strain-related reductions in oxidative stress.

Cause and effect relationship between myocardial mast cell number and matrix metalloproteinase activity.

The objectives of this study were to investigate the temporal response of left ventricular (LV) matrix metalloproteinase (MMP) activity and collagen volume fraction (CVF) induced by an aortocaval fistula and the role of cardiac mast cells in regulating MMP activity. LV tissue was analyzed for MMP activity, CVF, and mast cell number in rats euthanized at 0.5, 1, 2, 3, 5, 14, 21, 35, and 56 days. Additional rats treated with the mast cell membrane-stabilizing drug cromolyn sodium were euthanized 1, 2, and 3 days postfistula. Marked increases in MMP activity occurred rapidly and remained significantly elevated for 5 days before returning toward normal. A significant decrease in CVF occurred by day 5, but thereafter CVF rebounded to normal or above normal values. The number of myocardial mast cells also significantly increased postfistula, and there was a close association between mast cell density and MMP activity. Cromolyn treatment prevented the increase in mast cell number and MMP activity. Thus it is concluded that cardiac mast cells play a major role in the regulation of MMP activity.

Matrix metalloproteinase inhibition limits arterial enlargements in a rodent arteriovenous fistula model.

BACKGROUND: We administered a specific, nonselective matrix metalloproteinase (MMP) inhibitor (RS-113,456) to examine the effect of MMP inhibition on flow-mediated arterial enlargement in a rodent arteriovenous fistula (AVF) model. METHODS: Four groups of male Sprague-Dawley rats were created: sham (sham operated; n = 10), control (2.0 mm left common femoral AVF alone; n = 16), vehicle (AVF plus 0.5 mL vehicle orally twice a day; n = 20), and treatment (AVF plus 25 mg/kg RS-113,456 in 0.5 mL vehicle orally twice a day; n = 16). Heart rate, mean arterial pressure, and body weight were recorded on postoperative days 0, 7, 14, and 21. On day 21, AVF patency was confirmed, the infrarenal aorta and common iliac arteries were exposed, blood flow velocity and external diameter were measured, and wall shear stress (WSS) was calculated. Analysis was performed by paired, two-tailed Student t test, one-way analysis of variance, and the Bonferroni/Dunn procedure for post hoc testing. RESULTS: Heat rate, mean arterial pressure, and weight did not vary at any time between groups. Aortic and left iliac diameter was larger in the AVF groups than in sham groups (P < .001), and control and vehicle groups were larger than treatment groups (P < .0001). Changes in aortic and left iliac flow were also significant (AVF was more than sham and control, and vehicle was more than treatment). No difference in aortic and left iliac artery velocity and WSS or right iliac diameter, velocity, flow, or WSS was observed between groups. CONCLUSIONS: MMP inhibition diminishes flow-mediated arterial enlargement in the rat AVF model.

Role of neutral endopeptidase 24.11 in AV fistular rat model of heart failure.

OBJECTIVE: The aortovenocaval fistular (AVF) rat represents a model of heart failure caused by increased cardiac volume overload and reduced renal function. Both circulating vasoconstrictors like the renin-angiotensin-aldosterone system and vasodilators like atrial and brain natriuretic peptides (ANP and BNP) are activated in this animal model of heart failure. In addition, neutral endopeptidase 24.11 (NEP) in plasma and urine is elevated in AVF rats. In the present investigation we examined the renal and hormonal effects of the NEP inhibitor, ecadotril, in acute and chronic studies in rats with an aortovenocaval fistula (AVF). METHODS: Sprague Dawley rats (350-430 g) were prepared by introducing a shunt between abdominal aorta and the vena cava. RESULTS: Acute administration of the neutral endopeptidase inhibitor, ecadotril (30 mg/kg p.o.), significantly improved the reduced renal excretion of sodium in AVF rats (83 +/- 10 to 145 +/- 14 mumol/kg/h, P < 0.01) but had no significant effect in sham-operated rats. However, neutral endopeptidase activity in urine was significantly decreased after ecadotril in both groups. Plasma ANP was increased after ecadotril only in AVF rats (275 +/- 83 to 748 +/- 187 pg/ml, P < 0.05), whereas the increase in plasma BNP was not statistically significant. After 4 weeks of observation the ANP and BNP plasma levels, renin activity (PRA), angiotensin I, and neutral endopeptidase activity were significantly higher in AVF rats than in sham-operated rats. Four weeks on ecadotril (30 mg/kg p.o., b.i.d.) increased plasma ANP (245 +/- 48 as opposed to 450 +/- 77 pg/ml, P < 0.05) and decreased PRA (11.3 +/- 1.5 as opposed to 6.8 +/- 1.2 ng/ml/h, P < 0.005) in AVF rats. Plasma NEP activity was inhibited in both groups. Ventricle weight was significantly higher in AVF rats than in sham-operated controls, and ecadotril treatment over 4 weeks decreased ventricular hypertrophy to a slight extent. CONCLUSION: These results indicate that in the AVF rat model of heart failure the neutral endopeptidase inhibitor, ecadotril, improves the reduced kidney function in AVF rats by raising natriuretic peptides in plasma and probably in urine. NEP inhibition with ecadotril could therefore offer useful therapeutic possibilities in the treatment of heart failure.