Untreated Arteries Research Articles

Delivery of sirolimus with intravascular ultrasound to prevent vascular restenosis

Restenosis is the re-narrowing of arteries. It typically occurs after angioplasty treatment for peripheral artery diseasesand results in the need for invasive surgical treatments or amputation of the limb. Sirolimus is a known anti-restenosis drug, yet it requires long exposure time in the artery despite being quickly inactivated in blood. As such, there is a need for a targeted drug delivery methodology to promote slow-releasing sirolimus drug delivery vehicles. Our previous work suggested that sirolimus can be loaded into sound sensitive microparticles (SSMPs) comprised of PLGA. Here, we seek to determine if a bespoke intravascular device (IVUS) can deliver SSMPs to the arterial wall of a porcine artery in vivo. In this study, porcine arteries of three pigs were occluded by a balloon on the IVUS device distal to the treatment site, while drug loaded microparticles were injected through a sheath prior to ultrasound emission to facilitate uptake. We observed that the presence of SSMPs was larger in the arterial walls compared the blood and untreated arteries. The IVUS device provided a marginal increase in SSMPs in the treated arteries for most of the pigs. Thus, the in vivo studies suggested that the IVUS parameters were not yet optimized.

Pentagalloyl glucose (PGG) partially prevents arterial mechanical changes due to elastin degradation.

Elastic fibers are composed primarily of the protein elastin and they provide reversible elasticity to the large arteries. Degradation of elastic fibers is a common histopathology in aortic aneurysms. Pentagalloyl glucose (PGG) has been shown to bind elastin and stabilize elastic fibers in some in vitro studies and in vivo models of abdominal aortic aneurysms, however its effects on native arteries are not well described. Perform detailed studies of the biomechanical effects of PGG on native arteries and the preventative capabilities of PGG for elastin degraded arteries. We treated mouse carotid arteries with PGG, elastase (ELA), and PGG+ELA and compared the wall structure, solid mechanics, and fluid transport properties to untreated (UNT) arteries. We found that PGG alone decreased compliance compared to UNT arteries, but did not affect any other structural or biomechanical measures. Mild (30 sec) ELA treatment caused collapse and fragmentation of the elastic lamellae, plastic deformation, decreased compliance, increased modulus, and increased hydraulic conductance of the arterial wall compared to UNT. PGG+ELA treatment partially protected from all of these changes, in particular the plastic deformation. PGG mechanical protection varied considerably across PGG+ELA samples and appeared to correlate with the structural changes. Our results provide important considerations for the effects of PGG on native arteries and a baseline for further biomechanical studies on preventative elastic fiber stabilization.

Activation of protein kinase C impairs endothelium‐dependent vasorelaxation in isolated human omental resistance arteries

Chronic diseases such as obesity and diabetes are associated with endothelial dysfunction and increased cardiovascular disease risk. Notably, protein kinase C (PKC) has been identified as a major molecular contributor to chronic disease‐associated microvascular impairments. However, the molecular mechanisms by which PKC activation causes microvascular endothelial dysfunction are not fully understood. Herein, we tested the hypothesis that PKC activation impairs microvascular vasorelaxation through an endothelial nitric oxide synthase (eNOS)‐dependent mechanism in human isolated resistance arteries and cultured endothelial cells. Accordingly, resistance arteries were isolated from the omentum of 32 patients (52±3 years of age, 43.3±1.7 BMI, 76% women) that underwent abdominal surgery and analyzed for vasomotor function ex vivo using wire myography. We found that pharmacological inhibition of PKC (ruboxistaurin, 1μM) for 30 minutes enhanced endothelium‐dependent vasorelaxation in response to bradykinin relative to untreated arteries (p<0.05). This effect of ruboxistaurin was abrogated by NOS inhibition (L‐NAME, 300μM), suggesting that improved endothelium‐dependent relaxation with PKC antagonism is nitric oxide‐dependent (p<0.05). Next, we found that treatment with the PKC activator, phorbol 12‐myristate 13‐acetate (PMA, 0.5μM), for 30 minutes impaired endothelium‐dependent and ‐independent vasorelaxation, which was abolished with concomitant incubation of ruboxistaurin (p<0.05). Furthermore, we found that PMA treatment reduced Akt and eNOS activation in cultured endothelial cells (HUVEC; p<0.05). In conclusion, these findings suggest that in isolated human abdominal adipose tissue resistance arteries, PKC activation impairs microvascular endothelial function likely through an eNOS‐dependent mechanism. Follow‐up experiments are ongoing to determine the involvement of specific PKC isoforms and upstream molecular regulators contributing to PKC‐mediated impairments in endothelial function.Support or Funding InformationResearch support: National Institutes of Health grants: R01 HL137769 (JP), R01 HL088105 (LM‐L).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Coronary Vascular Effects of Leptin and Calpastatin in Lean vs. Obese Hearts

Recent evidence suggests a role for leptin and calpastatin in the pathogenesis of obesity‐induced coronary vascular dysfunction. However, how these factors affect coronary smooth muscle reactivity in lean vs. obese hearts is not well defined. Coronary arteries from lean and obese Ossabaw swine were isolated, cleaned of all surrounding adipose tissue, and incubated with or without leptin (3 – 30 ng/mL) or calpastatin (10 μM) for 30 min. Arteries were then pre‐constricted with the thromboxane mimetic U46619 (1μM). In untreated arteries, vasodilation to adenosine (0.01 – 30 μM) was decreased ~25% in obese compared to lean arteries. Administration of leptin dose‐dependently reduced vasodilation to adenosine, with the 30 ng/mL concentration attenuating dilation ~25% in both lean and obese arteries. In contrast, the presence of calpastatin impaired responses to adenosine (10 μM) in lean (~26%), but not obese arteries. In additional studies, coronary arteries from lean swine were cultured at 37oC in the presence/absence of leptin (30 ng/mL) or calpastatin (10 μM) for 3 days and subsequently subjected to functional isometric tension studies. Relative to untreated cultured arteries, incubation with leptin augmented active tension development to KCl‐induced depolarization (30 mM) and diminished vasodilation in response to adenosine (10 μM). Incubation with calpastatin modestly reduced vasodilation to adenosine but did not influence active tension development to KCl. These findings indicate that leptin and calpastatin differentially influence vascular function and suggest that these factors could contribute to the augmented contractile, impaired vasodilator phenotype of the obese coronary circulation.

Flow increase is decisive to initiate angiogenesis in veins exposed to altered hemodynamics.

Exposing a vein to altered hemodynamics by creating an arteriovenous (AV) shunt evokes considerable vessel formation that may be of therapeutic potential. However, it is unclear whether the introduction of oscillatory flow and/or flow increase is decisive. To distinguish between these mechanical stimuli we grafted a femoral vein into the arterial flow pathway of the contralateral limb in rats creating an arterioarterial (AA) loop (n = 7). Alternatively, we connected the femoral artery and vein using the vein graft, whereby we created an AV-loop (n = 27). Vessel loops were embedded in a fibrin filled chamber and blood flow was measured by means of flow probes immediately after surgery (day 0) and 15 days after loop creation. On day 15, animals were sacrificed and angiogenesis was evaluated using μCT and histological analysis. Mean flow increased from 0.5 to 2.4 mL/min and was elevated throughout the cardiac cycle at day 0 in AV-loops whereas, as expected, it remained unchanged in AA-loops. Flow in AV-loops decreased with time, and was at day 15 not different from untreated femoral vessels or AA-loop grafts. Pulsatile flow oscillations were similar in AV-and AA-loops at day 0. The flow amplitude amounted to ~1.3 mL/min which was comparable to values in untreated arteries. Flow amplitude remained constant in AA-loops, whereas it decreased in AV-loops (day 15: 0.4 mL/min). A large number of newly formed vessels were present in AV-loops at day 15 arising from the grafted vein. In marked contrast, angiogenesis originating from the grafted vein was absent in AA-loops. We conclude that exposure to substantially increased flow is required to initiate angiogenesis in grafted veins, whereas selective enhancement of pulsatile flow is unable to do so. This suggests that indeed flow and most likely wall shear stress is decisive to initiate formation of vessels in this hemodynamically driven angiogenesis model.

Intracoronary Infusion of Mononuclear Bone Marrow-Derived Stem Cells is Associated with a Lower Plaque Burden After Four Years

To assess the long-term effects of intracoronary stem cell (SC) infusion following acute myocardial infarction (AMI) on the local atherosclerotic process at the site of infusion after four years of follow-up. We evaluated 18 post-AMI patients. Group 1 consisted of nine patients given an intracoronary injection of autologous mononuclear SC, and group 2 consisted of nine patients given a placebo injection. The plaque count, plaque burden (PB) and coronary calcium score (CCS) were assessed using multislice 64 CT angiography of the coronary tree four years after SC injection. The total PB and CCS along the entire coronary segment distal to the site of infusion were significantly lower in the SC group (PB-702 mm(3) vs. 1.465 mm(3), p=0.0006; CCS-295 vs. 796, p＜ 0.0001).The mean PB per coronary artery was 112.12 mm(3)±16.82 mm(3) for the treated arteries vs. 189.56 mm(3)±20.98 mm(3) for the untreated arteries (p＜0.0001), while the mean CCS was 53.12±16.4 vs. 106.43±10.86 (p＜0.0001). In the SC group, there were significant differences in the mean PB (87.75 mm(3) vs. 112.12 mm(2), p= 0.005) and mean CCS (36.87 vs. 53.12, p=0.04) between the coronary segment infused with SC and the entire coronary artery. Multislice CT angiography of atheromatous plaques in the coronary tree four years after SC infusion in post-AMI patients indicates a significantly lower atheromatous plaque burden, as demonstrated by a lower plaque volume and lower CCS at the site of stem cell infusion compared with other coronary territories.

Beta radiation for renal nerve denervation: initial feasibility and safety

In small clinical trials, sympathetic renal denervation using radiofrequency (RF) energy shows promise in treating resistant hypertension. However, the RF procedure is lengthy and is associated with pain during ablation. Vascular brachytherapy, a proven treatment for in-stent restenosis, has the potential to cause nerve fibrosis. The purpose of the present study was to assess the safety and feasibility of renal artery brachytherapy for sympathetic renal denervation. A total of 10 normotensive domestic swine underwent vascular brachytherapy to left and right renal arteries using the Beta-Cath 3.5 Fr delivery system at doses of 25 Gy (n=8) and 50 Gy (n=8) at 2 mm from the source centre. These groups were compared to untreated arteries that served as control (n=4). Follow-up obtained at one or two months included angiogram, intravascular ultrasound, and histopathology analysis. The vascular brachytherapy procedure was safe and no apparent angiographic or ultrasound injuries to the vessel were seen. Histology showed a varying degree of thermal injury more pronounced in the 50 Gy group. The majority of examined nerves showed some degree of injury; there was a dose-related effect on nerve injury severity. There were varying degrees of arteriolar changes in the examined sections, with most showing a 2-20% degree of endothelial cell loss. This initial feasibility and safety study of renal nerve denervation, mediated by low and intermediate β-radiation dosages, indicates that this approach can cause nerve fibrosis while avoiding significant damage to the renal artery.

Impact of Local Endothelial Challenge with Cytomegalovirus or Glycoprotein B on Vasodilation in Intact Pressurized Arteries from Nonpregnant and Pregnant Mice1

Cytomegalovirus (CMV) infections are associated with vascular diseases in the human population. We have previously shown vascular dysfunction in systemic and uterine arteries dissected from nonpregnant (NP) mouse CMV (mCMV)-infected mice that was further impaired during late pregnancy (LP). CMV attachment alone through glycoprotein B (GB) can generate signals that impact vascular tone regulation. However, the contribution of direct virus interactions with endothelium to the vascular dysfunction we previously observed after in vivo mCMV infection is not known. We used a pressure myograph system to infuse GB or whole intact mCMV inside arteries dissected from uninfected mice and assessed vasodilation to methacholine infused inside pressurized arteries rather than applied abluminally. These results were compared to those observed after methacholine infusion into untreated arteries dissected from mCMV-infected mice. In mesenteric arteries, vasodilation to infused methacholine did not differ among treatments in NP or LP groups in contrast to previously published studies. However, increased vasoconstrictor activity was unmasked after blocking thromboxane receptors or prostaglandin production. Vasodilation in uterine arteries from uninfected NP mice to infused methacholine was increased by both GB and whole intact mCMV pretreatment. Untreated uterine arteries from mCMV-infected NP mice showed even greater vasodilation. There was no effect of GB or whole intact mCMV pretreatment in uterine arteries from uninfected LP mice, whereas vasodilation to infused methacholine was reduced in untreated uterine arteries from mCMV-infected LP mice. CMV exerts direct effects on vascular function which should be considered during viral reactivation leading to viremia and during GB-based vaccine administration.

Protection of protease-activated receptor 2 mediated vasodilatation against angiotensin II-induced vascular dysfunction in mice

BackgroundUnder conditions of cardiovascular dysfunction, protease-activated receptor 2 (PAR2) agonists maintain vasodilatation activity, which has been attributed to increased cyclooxygenase-2, nitric oxide synthase and calcium-activated potassium channel (SK3.1) activities. Protease-activated receptor 2 agonist mediated vasodilatation is unknown under conditions of dysfunction caused by angiotensin II. The main purpose of our study was to determine whether PAR2-induced vasodilatation of resistance arteries was attenuated by prolonged angiotensin II treatment in mice. We compared the vasodilatation of resistance-type arteries (mesenteric) from angiotensin II-treated PAR2 wild-type mice (WT) induced by PAR2 agonist 2-furoyl-LIGRLO-amide (2fly) to the responses obtained in controls (saline treatment). We also investigated arterial vasodilatation in angiotensin II-treated PAR2 deficient (PAR2-/-) mice.Results2fly-induced relaxations of untreated arteries from angiotensin II-treated WT were not different than saline-treated WT. Treatment of arteries with nitric oxide synthase inhibitor and SK3.1 inhibitor (L-NAME + TRAM-34) blocked 2fly in angiotensin II-treated WT. Protein and mRNA expression of cyclooxygenase-1 and -2 were increased, and cyclooxygenase activity increased the sensitivity of arteries to 2fly in only angiotensin II-treated WT. These protective vasodilatation mechanisms were selective for 2fly compared with acetylcholine- and nitroprusside-induced relaxations which were attenuated by angiotensin II; PAR2-/- were protected against this attenuation of nitroprusside.ConclusionsPAR2-mediated vasodilatation of resistance type arteries is protected against the negative effects of angiotensin II-induced vascular dysfunction in mice. In conditions of endothelial dysfunction, angiotensin II induction of cyclooxygenases increases sensitivity to PAR2 agonist and the preserved vasodilatation mechanism involves activation of SK3.1.

Small Vessel Replacement by Human Umbilical Arteries With Polyelectrolyte Film-Treated Arteries: In Vivo Behavior

The aim of this study was to evaluate the patency of human umbilical arteries treated with polyelectrolyte multilayers (PEMs) after rabbit implantation. The development of small-caliber vascular substitutes with high patency after implantation remains a real challenge for vascular tissue engineering. Cryopreserved human umbilical arteries were enzymatically de-endothelialized and the luminal surfaces were coated with poly(styrene sulfonate)/poly(allylamine hydrochloride) (PSS/PAH) multilayers. The PEM-untreated arteries and PEM-treated rabbit carotids were used as graft control. The native rabbit carotids were bypassed by grafts. The Doppler ultrasound evaluation, performed in vivo, showed that all PEM-treated grafts remained patent during the full experimental period, whereas after only 1 week, no blood circulation was detected in untreated arteries. Scanning electron microscopy and histological graft examination showed pervasive thrombus formation on the luminal surface of untreated arteries after 1 week and clean luminal surface for treated arteries for at least up to 12 weeks. The arterial wall cells were identified through alpha-smooth muscle actin alphaupsilondelta platelet endothelial cell adhesion molecule-1 expression. The smooth muscle cells positive to alpha-smooth muscle actin were identified in adventitia and media and the endothelial cells positive to platelet endothelial cell adhesion molecule in intima. Von Kossa reaction didn't reveal any calcium salt deposits on the wall arteries, suggesting a good wall remodelling with no sign of graft rejection. The in vivo evaluation of human umbilical arteries treated with PSS/PAH multilayers demonstrated a high graft patency after 3 months of implantation. Such modified arteries could constitute a useful option for small vascular replacement.

Oxyhemoglobin-Induced Expression of R-Type Ca 2+ Channels in Cerebral Arteries

Cerebral vasospasm after subarachnoid hemorrhage (SAH) is a major contributor to mortality and morbidity after aneurysm rupture. Recently, R-type voltage-dependent Ca(2+) channel (VDCC) expression has been associated with increased cerebral artery constriction in a rabbit model of SAH. The goal of the present study was to examine whether the blood component oxyhemoglobin (oxyHb) can mimic the ability of SAH to cause R-type VDCC expression in the cerebral vasculature. Rabbit cerebral arteries were organ cultured in serum-free media for up to 5 days in the presence or absence of purified oxyHb (10 micromol/L). Diameter changes in response to diltiazem, (L-type VDCC antagonist) and SNX-482 (R-type VDCC antagonist) were recorded at day 1, 3, or 5 in arteries constricted by elevated extracellular potassium. RT-PCR was performed on RNA extracted from arteries cultured for 5 days (+/-oxyHb) to assess VDCC expression. After 5 days, oxyHb-treated arteries were less sensitive and partially resistant to diltiazem compared to similar arteries organ cultured in the absence of oxyHb. Further, SNX-482 dilated arteries organ cultured for 5 days in the presence, but not in the absence, of oxyHb. RT-PCR revealed that oxyHb treated arteries expressed R-type VDCCs (Ca(V) 2.3) in addition to L-type VDCCs (Ca(V) 1.2), whereas untreated arteries expressed only Ca(V) 1.2. These results demonstrate that oxyhemoglobin exposure for 5 days induces the expression of Ca(V) 2.3 in cerebral arteries. We propose that oxyhemoglobin contributes to enhanced cerebral artery constriction after SAH via the emergence of R-type VDCCs.

EDHF prevents endothelial dysfunction in response to a high‐fat diet in the Wistar rat

Individuals consuming a high‐fat diet, often display reduced responsiveness to endothelial‐dependant vasodilators such as acetylcholine or bradykinin. Endothelium‐derived hyperpolarizing factor (EDHF) may be an important vasoactive factor in resistance arteries from many vascular beds. However, its importance relative to other endothelial derived relaxing factors such as nitric oxide and prostaglandins is unclear. Although, genetic models of obesity and type II diabetes have demonstrated that EDHF‐mediated vasodilation is impaired; it is unknown if EDHF function is also affected by a high‐fat diet. 3–4 wk old Male Wistar rats were fed either a high‐fat diet (60% lard) or chow for 10–11 wks. Isolated small mesenteric arteries were pre‐constricted with Phenylephrine (PE; 10−9 – 10−5 M) and administered acetylcholine (ACh; 10−8 – 10−6 M) in the presence of either vehicle or Indomethacin (10 μm) and N(ω)‐nitro‐L‐arginine (L‐NNA; 100 μm). Vasoconstrictor responses to PE were also assessed in a separate set of untreated arteries. Blood pressure was determined by tail‐cuff. Body and epididymal fat pad weight were significantly greater in fat‐fed rats compared to chow fed. Mean arterial blood pressure was not different between groups. ACh‐induced dilation in arteries treated with vehicle was similar between chow and fat‐fed rats. Pre‐treating arteries with Indomethacin and L‐NNA blunted ACh‐mediated vasodilation in chow fed rats but did not alter ACh responses in arteries from fat‐fed animals. Reactivity to PE was not different between groups. These results suggest that role for EDHF in ACh‐mediated vasodilation is enhanced in small mesenteric arteries following a high‐fat diet.

Endogenous adipose tissue as a hemostatic: Use in microsurgery

Bleeding is a frequent complication of microsurgical repair of small blood vessels and time is spent while hemostasis is accomplished. We studied the hemostatic effect of endogenous adipose tissue on bleeding from rat femoral arterial anastomoses. We measured bleeding time (time from removal of clamps to cessation of active bleeding) and mean arterial blood velocity (using a micro-Doppler system), the latter immediately after anastomosis, and again 7 days post-anastomosis. Bleeding time for vessels with fat applied to the artery was 50% less than when no fat was applied. Blood velocity by day 7 post-anastomosis returned to values equivalent to those for intact arteries. Histological evaluation of the anastomotic site demonstrated no significant differences in inflammatory response between fat-treated and untreated arteries. These data suggest that endogenous adipose tissue may be a useful hemostatic agent devoid of significant effects on small artery blood velocity or histology.

Abstract 240: Inhibition of Glycogen Synthase Kinase 3 Beta Improves EPC Yield, Function and Ameliorates Arterial Repair Post-injury and Stenting

Endothelial progenitor cells (EPCs) are circulating undifferentiated pluripotent cells capable of reconstituting endothelium in vivo and diminishing lesion formation following arterial injury. We tested the hypothesis that treatment with a specific soluble GSK 3β-inhibitor (GSK3βi) would improve the in vitro yield and function of EPCs from patients with coronary artery disease (CAD) and augment their in vivo potential to improve arterial repair after injury and stent implantation. In vitro treatment with GSK3βi at 1× (IC50) and 2× (IC50) increased the number of cells that dual labelled for acLDL and UEA-1 lectin on day 7 (13.4±3.2 vs. 39.9±5.3 vs. 43.8±10.1; p < 0.05). Furthermore, GSK3βi significantly augmented CAD-EPC survival to levels equal to those of healthy controls, increased VEGF secretion (7.1±1.6 vs 30.0±8.9, p<0.05) and resulted in a 4× increase in adhesion (p<0.05). Using Q-PCR, mRNA copy numbers for VLA-1,2,3,4 and 5 were assayed; however, only VLA-4 levels increased (4×, p<0.01). Use of a VLA-4 specific blocking antibody confirmed that the improved EPC adhesion was specifically mediated by VLA-4. Finally, the effects of GSK 3β inhibition were tested in two in vivo models of arterial injury. CD-1 athymic nude mice underwent femoral artery wire injury and injection of CAD-EPCs that were either GSK3βi treated, untreated, or injection with vehicle alone. GSK3βi treated EPCs improved the ability of xenotransplanted cells to inhibit lesion formation when compared to untreated and vehicle arteries respectively (intima/media ratios: 0.3±0.1 vs. 5.3±1.2 vs 11.2±2.7 μm, p<0.01). GSK3βi coated stents were inserted into rabbit carotid arteries. Fourteen days after stent deployment, GSK3βi stents resulted in a 44% increase in re-endothelialization (assessed by scanning electron microscopy and compared to vehicle coated stents; p<0.01) and a 16% trend towards a reduction in neointima formation (p<0.09). In summary, our data demonstrates that GSK3βi improves EPC function in vitro and ameliorates arterial repair following injury and stent insertion in vivo .

Dexamethasone Alters Vascular Reactivity by Enhancing COX-Related Vasodilatation in Fetal Ovine Carotids

Based on preliminary studies that contractile efficacy was altered in vertebral and basilar arteries from neonatal donors treated with postnatal glucocorticoids, we examined the hypothesis that postnatal dexamethasone (DEX), a glucocorticoid used for respiratory disease in neonates, can alter vascular reactivity. Using near-term fetal lamb carotids, we measured 5-hydroxytryptamine (5-HT) dose-response relationship in DEX-treated and untreated arteries. We found that DEX incubation for 1 h had no effect on 5-HT sensitivity and agonist affinity but significantly reduced 5-HT contractile efficacy, a response that became even more pronounced after 4 h of DEX treatment. Coincubation of DEX-treated arteries with INDO for 4 h reversed this DEX-induced attenuation in 5-HT contractile efficacy, although DEX had no significant effects on cyclooxygenase (COX)-1 and COX-2 protein abundance. This data suggests that DEX alters vascular reactivity through a COX-related mechanism, with possible repercussions to neurological injury.

Role of Endogenous Fas (CD95/Apo-1) Ligand in Balloon-Induced Apoptosis, Inflammation, and Neointima Formation

Fas (CD95/Apo-1) ligand (FasL)-induced apoptosis in Fas-bearing cells is critically involved in modulating immune reactions and tissue repair. Apoptosis has also been described after mechanical vascular injury such as percutaneous coronary intervention. However, the relevance of cell death in this context of vascular repair remains unknown. To determine whether FasL-induced apoptosis is causally related to neointimal lesion formation, we subjected FasL-deficient (generalized lymphoproliferative disorder [gld], C57BL/6J) and corresponding wild-type (WT) mice to carotid balloon distension injury, which induces marked endothelial denudation and medial cell death. FasL expression in WT mice was induced in injured vessels compared with untreated arteries (P<0.05; n=5). Conversely, absence of functional FasL in gld mice decreased medial and intimal apoptosis (terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling [TUNEL] index) at 1 hour and 7 days after balloon injury (P<0.05; n=6). In addition, peritoneal macrophages isolated from gld mice showed no apoptosis and enhanced migration (P<0.05; n=4). In parallel, we observed increased balloon-induced macrophage infiltrations (anti-CD68) in injured arteries of FasL-deficient animals (P<0.05; n=6). Together with enhanced proliferation (bromodeoxyuridine index; P<0.05), these events resulted in a further increase in medial and neointimal cells (P<0.01; n=8) with thickened neointima in gld mice (intima/media ratio, x3.8 of WT; P<0.01). Our data identify proapoptotic and antiinflammatory effects of endogenous FasL as important factors in the process of neointimal lesion formation after balloon injury. Moreover, they suggest that activation of FasL may decrease neointimal thickening after percutaneous coronary intervention.

Decreased alpha‐2B‐adrenoceptor (α 2B ‐AR) expression augments nitric oxide (NO) dependent dilation

Mice heterozygous for a non-functional α2B-AR (Het) are more susceptible to NO synthase-inhibition hypertension than wild type (WT) mice. Furthermore, Het mice have augmented NO-dependent vasodilation and increased vascular expression of endothelial NOS (eNOS). We hypothesized that this increased eNOS inhibits both myogenic and agonist-stimulated tone in arteries from Het mice. In isolated, pressurized mesenteric arteries (inner diameter = 170±3 μm), myogenic tone was not different between groups in untreated arteries. NOS-inhibition (L-NNA, 10−4 mol/L) increased myogenic tone more in Het arteries than in WT arteries PE constriction of mesenteric arteries. * different between groups. # (15±3% Het versus 5±3% WT; p<0.05). Phenylephrine (PE, 10−5 mol/L) constricted Het arteries less than WT arteries (Figure). However, in L-NNA treated arteries, PE constriction was not different between Het and WT arteries. In contrast, cyclooxygenase (COX) inhibition (indomethacin, 10−5 mol/L) decreased PE constriction only in WT arteries. COX inhibition was reversed by L-NNA, suggesting COX products inhibit NO production in WT but not Het arteries. Therefore arteries from Het mice (with decreased α2B-AR expression) have diminished myogenic and α1-AR-induced constriction that is apparently caused by increased NO-mediated dilation. In agreement with our previous observation that COX inhibition increases NO production in mesenteric arteries, the augmented NO dilation in Het arteries appears to be caused in part by loss of COX-dependent inhibition of NOS. Figure 1Open in figure viewerPowerPoint (Supported by AHA Established Investigator award and EPA RD-83186001, NLK)

Role of the PKC/CPI‐17 pathway in enhanced contractile responses of mesenteric arteries from diabetic rats to α‐adrenoceptor stimulation

Protein kinase C (PKC) may contribute to enhanced contractile responses of arteries from streptozotocin-diabetic rats to stimulation of G-protein coupled receptors. This was investigated by comparing the effects of PKC inhibitors on contractile responses of mesenteric arteries from diabetic and age-matched control rats to noradrenaline (NA) and endothelin-1 (ET-1). The effects of NA and ET-1 on the distribution of three isoforms of PKC implicated in contraction were also determined. In addition, the effect of NA on phosphorylation of CPI-17, a substrate for PKC, was investigated. Contractile responses of endothelium-denuded arteries from diabetic rats to NA were enhanced, but were normalized by PKC inhibition. In contrast, contractile responses to ET-1 were not significantly different, and were blocked to a similar extent by PKC inhibition, in arteries from control and diabetic rats.NA produced only a small increase in particulate levels of PKCepsilon in control arteries (to 125+/-8% of levels in untreated arteries), but a significant increase in particulate PKCalpha (to 190+/-22%) and a much greater increase in particulate PKCepsilon (to 230+/-19%) in arteries from diabetic rats. ET-1 increased particulate PKCalpha and epsilon to a similar extent in arteries from control and diabetic rats.NA significantly enhanced CPI-17 phosphorylation from a basal level of 22+/-10 to 71+/-7% of total in arteries from diabetic rats, and this was prevented by PKC inhibition. NA had no detectable effect on CPI-17 phosphorylation in arteries from control rats. These data suggest that NA-induced activation of PKC and CPI-17, its downstream target, is selectively enhanced in arteries from diabetic rats, and mediates the enhanced contractile responses to this agonist.

C-Type Natriuretic Peptide Inhibits Constrictive Remodeling Without Compromising Re-Endothelialization in Balloon-Dilated Renal Arteries

To investigate the long-term effect of local, liposome-mediated gene transfer of C-type natriuretic peptide (CNP) plasmid versus CNP protein on restenosis in porcine renal arteries following balloon angioplasty. The renal arteries of 15 pigs were dilated and the adventitia at the site of balloon injury injected with CNP protein, pCR3.1 plasmid encoding CNP, or the beta-galactosidase gene (control) via a needle injection catheter. Five animals receiving the CNP and control genes in dilated arteries were sacrificed after 3 weeks to analyze re-endothelialization, proliferation, and early CNP expression. Ten animals designated for the long-term experiments (3 months) were treated with the CNP gene versus CNP protein (n=3), the CNP gene versus the control gene (n=3), and the CNP protein versus the control gene (n=3). One animal served as a dilated non-treated control. Transfection and expression of CNP and beta-galactosidase were measured by polymerase chain reaction (PCR) and reverse transcription PCR. Renal arterial lumen narrowing was measured with angiography and histology. Endothelialization was assessed using Evans blue stain; vWF, CD31, factor VIII, and Ki67 were markers for immunohistochemical analysis. An intact endothelial layer was seen at 3 weeks following angioplasty in all transfected arteries. Three months following treatment, computer-assisted morphometric analysis revealed significant enlargement of the arterial cross-sectional areas in CNP plasmid- treated vessels compared to dilated but untreated arteries (CNP plasmid +34.8%+/-13.9% versus CNP protein -1.75%+/-19.9% versus beta-galactosidase -47.0%+/-13.9%, p<0.01). Angiographic analysis showed significant enlargement of the arterial diameter compared to dilated, untreated arteries (CNP plasmid +20.8%+/-6.8% versus CNP protein +5.7%+/-6.0% versus beta-galactosidase -24.5%+/-10.2%, p<0.01). Local application of CNP plasmid proved superior to CNP protein in producing rapid re-endothelialization and significantly enlarging the renal arterial lumen following dilation.

Native coronary disease progression exceeds failed revascularization as cause of angina after five years in the bypass angioplasty revascularization investigation (BARI)

Coronary angiograms obtained five years following revascularization were examined to assess the extent of compromise in myocardial perfusion due to failure of revascularization versus progression of native disease. The Bypass Angioplasty Revascularization Investigation (BARI) randomized revascularization candidates between bypass surgery and angioplasty. Entry and five-year angiograms from 407 of 519 (78%) patients at four centers were analyzed. Analysis of the distribution of coronary vessels and stenoses provided a measure of myocardial jeopardy that correlates with presence of angina. The extent to which initial benefits of revascularization were undone by failed revascularization versus native disease progression was assessed. Myocardial jeopardy fell following initial revascularization, from 60% to 17% for percutaneous coronary intervention (PCI)-treated patients compared with 60% to 7% for coronary artery bypass graft (CABG) surgery patients (p < 0.001), rebounding at five years to 25% for PCI and 20% for surgery patients (p = 0.01). Correspondingly, angina prevalence was higher at five years in PCI-treated patients than in surgery-treated patients (28% vs. 18%; p = 0.03). However, myocardial jeopardy at five years, and not initial treatment (PCI vs. surgery), was independently associated with late angina. Increased myocardial jeopardy from entry to five-year angiogram occurred in 42% of PCI-treated patients and 51% of CABG-treated patients (p = 0.06). Among the increases in myocardial jeopardy, two-thirds occurred in previously untreated arteries. Native coronary disease progression occurred more often than failed revascularization in both PCI- and CABG-treated patients as a cause of jeopardized myocardium and angina recurrence. These results support intensive postrevascularization risk-factor modification.