Pharmacokinetics and pharmacodynamics of the novel monobactam LYS228 in a neutropenic murine thigh model of infection.

Objectives: The neutropenic murine thigh infection model and a dose-fractionation approach were used to determine the pharmacokinetic/pharmacodynamic (PK/PD) relationship of LYS228, a novel monobactam antibiotic with activity against Enterobacteriaceae including carbapenem-resistant strains. Methods: Mice (n = 4 per group) were inoculated with Enterobacteriaceae strains via intramuscular injection. Two hours post-bacterial inoculation, treatment with LYS228 was initiated. Animals were euthanized with CO2 24 h after the start of therapy and bacterial counts (log10 cfu) per thigh were determined. PK parameters were calculated using free (f) plasma drug levels. Results: Following a dose-fractionation study, non-linear regression analysis determined that the predominant PK/PD parameter associated with antibacterial efficacy of LYS228 was the percentage of the dosing interval that free drug concentrations remained above the MIC (%fT>MIC). In a dose-dependent manner, LYS228 reduced the thigh bacterial burden in models established with Enterobacteriaceae producing ß-lactamase enzymes of all classes (e.g. ESBLs, NDM-1, KPC, CMY-2 and OXA-48). The range of the calculated static dose was 86-649 mg/kg/day for the isolates tested, and the magnitude of the driver of efficacy was 37-83 %fT>MIC. %fT>MIC was confirmed as the parameter predominantly driving efficacy as evidenced by a strong coefficient of determination (r2 = 0.68). Neutrophils had minimal impact on the effect of LYS228 in the murine thigh infection model. Conclusions: LYS228 is efficacious in murine thigh infection models using ß-lactamase-producing strains of Enterobacteriaceae, including those expressing metallo-ß-lactamases, ESBLs and serine carbapenemases, with the PK/PD driver of efficacy identified as %T>MIC.

Extracellular nucleotides affect pericyte-mediated regulation of rat in situ vasa recta diameter.

AIM: We hypothesized that extracellular nucleotides, established as being released from renal tubular epithelial cells, act at pericytes to regulate vasa recta capillary diameter. METHODS: A rat live kidney slice model and video imaging techniques were used to investigate the effects of extracellular nucleotides on in situ (subsurface) vasa recta diameter at pericyte and non-pericyte sites. In addition, RT-qPCR was used to quantify P2 receptor mRNA expression in isolated vasa recta. RESULTS: Extracellular ATP, UTP, benzylbenzyl ATP (BzATP) or 2-methylthioATP (2meSATP) evoked a significantly greater vasoconstriction of subsurface vasa recta at pericytes than at non-pericyte sites. The rank order of agonist potency was BzATP = 2meSATP > ATP = UTP. The vasoconstriction evoked at pericyte sites by ATP was significantly attenuated by the P2 receptor antagonists suramin, pyridoxal phosphate-6-azo(benzene-2,4-disulfonic acid) (PPADS) or Reactive Blue-2 (RB-2). UTP-evoked vasoconstriction at pericytes was attenuated by suramin or RB-2 but not PPADS. Interestingly, suramin or PPADS, when applied in the absence of a P2 receptor agonist, evoked a weak but significant vasoconstriction of vasa recta at pericyte sites, suggesting tonic vasodilation by nucleotides. Significant levels of P2X(1, 3 and 7) and P2Y(4 and 6) receptor mRNA were detected in vasa recta. CONCLUSION: Extracellular nucleotides act at pericytes to cause vasoconstriction of in situ vasa recta. Pharmacological characterization, supported by RT-qPCR data, suggests that P2X(1 and 7) and P2Y(4) receptors mediate nucleotide-evoked vasoconstriction of vasa recta by pericytes. We propose that nucleotides released from renal tubular epithelial cells, in close proximity to vasa recta capillaries, are key in regulating renal medullary blood flow.

A 5-year analysis of rapid response system activation at an in-hospital haemodialysis unit.

OBJECTIVE: To study the incidence, patient and event characteristics, and outcome of rapid response system (RRS) activation on an in-hospital haemodialysis unit. DESIGN: Retrospective review of all RRS events on an in-hospital 10-bed haemodialysis unit over a 64-month period (November 2001 to February 2007). SETTING: University of Pittsburgh Medical Center Presbyterian Hospital, a 730-bed academic, urban, tertiary care adult hospital in the USA. Interventions None. RESULTS: Over a 64-month-period, 107 of 8928 patients undergoing haemodialysis on the dialysis unit required an RRS activation (12 events/1000 patients dialysed). The most common reasons for RRS activation were respiratory distress/hypoxaemia (27%) and mental status change (24%). Predictors of in-hospital mortality included old age (33% in-hospital mortality for patients aged 65 years or older vs 14% for patients aged less than 65 years; χ(2) = 5.66, df = 1, p = 0.017), and RRS activation due to a respiratory abnormality (37% mortality for respiratory codes vs 18% for all other codes; χ(2) = 4.12, df = 1, p = 0.042). Surprisingly, only 71% of the patients who had an RRS event had the event as dialysis was occurring. Twenty-four patients (22%) met one or more RRS activation criteria upon first vital sign check in the dialysis unit; RRS was activated on 12 (11%) of these patients before dialysis was started. Nineteen (18%) additional patients had an RRS event after their dialysis session had ended, while awaiting transport back to their unit. CONCLUSIONS: From our findings, it can be suggested that critical events often occur before and after dialysis treatment, during or awaiting transport. Careful assessment of these high-risk patients before and after transport, to and from the dialysis unit may be warranted.

Analysis of major complications associated with arterial catheterisation.

INTRODUCTION: Arterial catheterisation is used for continuous haemodynamic monitoring in patients undergoing surgery and in critical care units. Although it is considered a safe procedure, a major complication such as arterial occlusion and limb gangrene can occur. OBJECTIVE: To determine the incidence, outcome and potential to avoid complications associated with arterial catheterisation. METHODS: The number of arterial catheterisation was determined using an anaesthesiology and critical care medicine billing database over a period of 4 years (1 January 2003 to 31 December 2006). Possible major complications were identified from two hospital databases; all identified charts were screened and then reviewed by an expert panel that determined causation. A major complication was defined as requiring operative intervention and/or resulting in permanent harm. RESULTS: 15 (0.084%) major complications were identified among 17 840 instances of arterial catheterisation insertions. Of 15 arterial catheterisations, nine were performed in the operating room and six in the intensive care unit. Nine patients suffered ischaemic injury, which progressed to gangrene in three patients. Three patients developed haematoma that required surgical evacuation; two of these required vascular repair. One patient had compartment syndrome requiring fasciotomy and two patients had sheared catheter fragments that needed to be removed. All 15 patients had multiple comorbidities, and those in the operating room had an American Society of Anesthesiologists score of >or=3. Seven (46.6%) had arterial catheterisation done under emergent circumstances. Six (40%) died during hospitalisation because of complications unrelated to arterial catheterisation. CONCLUSION: Arterial catheterisation had a very low rate of major complications. They seem associated with high severity of illness and emergency surgery.

Patient safety on the otolaryngology service: the role of an established rapid response system.

OBJECTIVE: To study the medical emergencies occurring on a tertiary otolaryngology service identified using a rapid response system (RRS). DESIGN: Retrospective chart review of RRS activations during 21 months. SETTING: Specialised otolaryngology care unit within the University of Pittsburgh Medical Center Presbyterian/Montefiore Hospital, a tertiary, academic, teaching hospital in the USA. INTERVENTION(S): None. RESULTS: 1171 unit admissions. Unit mortality was 5.1/1000 admissions. 53 patients were involved in 67 RRS activations (4/53 deaths). 32 of 67 events were due to respiratory derangements, most commonly pneumonia. 18 of 67 events were due to cardiovascular abnormalities, most commonly hypertension and myocardial infarction. 11 of 67 events were secondary to mental status changes, several of which were related to adverse drug events. 6 of 67 events were secondary to acute bleeding. 23 of 67 events occurred within 24 h of patient transfer/admission, 14 of those after operations. RRS activation was a marker for in-hospital death (RR 42.2, 95% CI 7.9 to 225.2) compared with that in patients not activating the RRS. CONCLUSIONS: Although otolaryngology care units attempt to prevent adverse events, emergencies still occur. RRSs identify deteriorating otolaryngology patients who are at increased risk for mortality. RRSs are an efficient mechanism of intervention during a medical emergency. RRSs provide a convenient method of identifying medical/system errors and educational opportunities.

Monomeric inducible nitric oxide synthase localizes to peroxisomes in hepatocytes.

Hepatocytes are capable of repeated inducible NO synthase (iNOS) expression, which occurs under inflammatory and stress conditions. This iNOS expression regulates a number of cellular functions as well as cell viability. To better understand the posttranslational mechanisms that regulate the fate of iNOS in these cells, we characterized the iNOS distributed within peroxisomes. The selective permeabilization of membranes (plasma vs. peroxisomal) confirmed that there are cytosolic and peroxisomal pools of iNOS in cytokine-stimulated hepatocytes and that the iNOS protein associates with peroxisome. Detergent solubilization of the membrane fraction released iNOS to the soluble fraction. iNOS localized to membrane fraction is predominantly monomeric, but dimerization is partially reconstituted rapidly upon incubation with tetrahydrobiopterin. The reconstituted iNOS exhibits a lower specific activity than iNOS isolated from the soluble pool. Depletion of intracellular tetrahydrobiopterin with an inhibitor of de novo pterin synthesis resulted in a predominance of monomeric iNOS without a greater relative distribution of iNOS to the peroxisomal pool. Thus, iNOS exists in a least two pools in hepatocytes: a soluble pool composed of both active dimer and monomer and a peroxisomal pool of monomeric iNOS. iNOS might localize to peroxisomes in long-lived cells such as hepatocytes as a protective mechanism to remove incompetent enzyme.

Use of medical emergency team responses to reduce hospital cardiopulmonary arrests.

BACKGROUND: Medical emergency team (MET) responses have been implemented to reduce inpatient mortality, but data on their efficacy are sparse and there have been no reports to date from US hospitals. OBJECTIVES: To determine how the incidence and outcomes of cardiac arrests have changed following increased use of MET. METHODS: Objective criteria for MET activation were created and disseminated as part of a crisis management program, after which there was a rapid and sustained increase in the use of MET. A retrospective analysis of clinical outcomes was performed to compare the incidence and mortality of cardiopulmonary arrest before and after the increased use of MET. RESULTS: A retrospective analysis of 3269 MET responses and 1220 cardiopulmonary arrests over 6.8 years showed an increase in MET responses from 13.7 to 25.8 per 1000 admissions (p<0.0001) after instituting objective activation criteria. There was a coincident 17% decrease in the incidence of cardiopulmonary arrests from 6.5 to 5.4 per 1000 admissions (p = 0.016). The proportion of fatal arrests was similar before and after the increase in use of MET. CONCLUSIONS: Increased use of MET may be associated with fewer cardiopulmonary arrests.

Use of medical emergency team (MET) responses to detect medical errors.

BACKGROUND: No previous studies have investigated whether medical emergency team (MET) responses can be used to detect medical errors. OBJECTIVES: To determine whether review of MET responses can be used as a surveillance method for detecting medical errors. METHODS: Charts of all patients receiving MET responses during an 8 month period were reviewed by a hospital based Quality Improvement Committee to establish if the clinical deterioration that prompted the MET response was associated with a medical error (defined as an adverse event that was preventable with the current state of medical knowledge). Medical errors were categorized as diagnostic, treatment, or preventive errors using a descriptive typology based on previous published reports. RESULTS: Three hundred and sixty four consecutive MET responses underwent chart review and 114 (31.3%) were associated with medical errors: 77 (67.5%) were categorized as diagnostic errors, 68 (59.6%) as treatment errors, and 30 (26.3%) as prevention errors. Eighteen separate hospital care processes were identified and modified as a result of this review, 10 of which involved standardization. CONCLUSIONS: MET review may be used for surveillance to detect medical errors and to identify and modify processes of care that underlie those errors.

Clostridium difficile colitis in patients after kidney and pancreas-kidney transplantation.

Limited data exist about Clostridium difficile colitis (CDC) in solid organ transplant patients. Between 1/1/99 and 12/31/02, 600 kidney and 102 pancreas-kidney allograft recipients were transplanted. Thirty-nine (5.5%) of these patients had CDC on the basis of clinical and laboratory findings. Of these 39 patients, 35 have information available for review. CDC developed at a median of 30 days after transplantation, and the patients undergoing pancreas-kidney transplantation had a slightly higher incidence of CDC than recipients of kidney alone (7.8% vs. 4.5%, P>0.05). All but one patient presented with diarrhea. Twenty-four patients (64.9%) were diagnosed in the hospital, and CDC occurred during first hospitalization in 14 patients (40%). Treatment was with oral metronidazole (M) in 33 patients (94%) and M+oral vancomycin (M+V) in 2 patients. Eight patients had recurrent CDC, which occurred at a median of 30 days (range 15-314) after the first episode. Two patients (5.7%) developed fulminant CDC, presented with toxic megacolon, and underwent colectomy. One of them died; the other patient survived after colectomy. CDC should be considered as a diagnosis in transplant patients with history of diarrhea after antibiotic use, and should be treated aggressively before the infection becomes complicated.

Antioxidant enzymes suppress nitric oxide production through the inhibition of NF-kappa B activation: role of H(2)O(2) and nitric oxide in inducible nitric oxide synthase expression in macrophages.

Reactive molecules O(-)(2), H(2)O(2), and nitrogen monoxide (NO) are produced from macrophages following exposure to lipopolysaccharide (LPS) and involved in cellular signaling for gene expression. Experiments were carried out to determine whether these molecules regulate inducible nitric oxide synthase (iNOS) gene expression in RAW264.7 macrophages exposed to LPS. NO production was inhibited by the antioxidative enzymes catalase, horseradish peroxidase, and myeloperoxidase but not by superoxide dismutase (SOD). In contrast, the NO-producing activity of LPS-stimulated RAW264.7 cells was enhanced by the NO scavengers hemoglobin (Hb) and myoglobin. The antioxidant enzymes decreased levels of iNOS mRNA and protein in LPS-stimulated RAW264.7 cells, whereas the NOS inhibitor N(G)-monomethyl-L-arginine as well as Hb increased the level of iNOS protein but not mRNA, indicating that NO inhibits iNOS protein expression. NF-kappa B was activated in LPS-stimulated RAW264.7 cells and the activation was significantly inhibited by antioxidant enzymes, but not by Hb. Similar results were obtained using LPS-stimulated rodent peritoneal macrophages. Extracellular O(-)(2) generation by LPS-stimulated macrophages was suppressed by SOD, but not by antioxidative enzymes, while accumulation of intracellular reactive oxygen species was inhibited by antioxidative enzymes, but not by SOD. Exogenous H(2)O(2) induced NF-kappa B activation in macrophages, which was inhibited by catalase and pyrroline dithiocarbamate (PDTC). H(2)O(2) enhanced iNOS expression and NO production in peritoneal macrophages when added with interferon-gamma, and the effect of H(2)O(2) was inhibited by catalase and PDTC. These findings suggest that H(2)O(2) production from LPS-stimulated macrophages participates in the upregulation of iNOS expression via NF-kappa B activation and that NO is a negative feedback inhibitor of iNOS protein expression.

Nitric oxide induces murine thymocyte apoptosis by oxidative injury and a p53-dependent mechanism.

Previously, we showed that NO induces thymocyte apoptosis via a caspase-1-dependent mechanism [(1) ]. In the present study, we investigated the role of heme oxygenase, catalase, bax, and p53 in this process. The NO donor, S-nitroso-N-acetyl penicillamine (SNAP), induced DNA fragmentation in thymocytes in a time- and concentration-dependent way. SNAP (100 microM) induced 50--60% apoptosis; higher doses did not increase the rate of apoptosis significantly. SNAP decreased catalase and heme iron (Fe) levels without affecting superoxide dismutase, glutathione, or total Fe stores in thymocytes. SNAP significantly increased the expression of heme oxygenase 1 (HSP-32), p53, and bax but not bcl-2. Treatment with the heme oxygenase inhibitor, tin protoporphyrin IX inhibited SNAP-induced thymocyte apoptosis. Furthermore, thymocytes from p53 null mice were resistant to NO-induced apoptosis. Our data suggest that NO may induce its cytotoxic effects on thymocytes by modulating heme oxygenase and catalase activity as well as up-regulating pro-apoptotic proteins p53 and bax.

Effect of plasma and matrix proteins on defensin-induced impairment of phagocytic killing by adherent neutrophils.

Infection is too often associated with prosthetic devices. Increased susceptibility to infection at these surgical sites appears to be associated with defective local phagocytic killing. The mechanisms for neutrophil down-regulation, however, continue to be obscure. We have recently demonstrated that cytotoxic substances are released from granulocytes associated with materials. One group of releasants, the cationic human neutrophil peptide(s) (also called defensins) not only impairs the antimicrobial capacity of the granulocyte that releases it but also impairs bystander phagocytes. Because plasma or matrix proteins soon become associated with implants, we investigated the interactive effect of adding these proteins, singly and in combination, on the microbicidal effect of bystander cells. Some plasma/matrix proteins (whole plasma, albumin, fibrinogen, and fibronectin) strongly interfered with the anti-microbicidal effects generated by neutrophil-polystyrene interaction. Other proteins (vitronectin and laminin) were without effect. These results suggest that protein composition at the prosthetic implant site could have a significant effect on infectivity, depending on whether neutrophils releasants were attenuated. In the absence of attenuation, the local environment would be hostile to host defenses, permitting bacterial survival and proliferation.

Nitric oxide protects thymocytes from gamma-irradiation-induced apoptosis in correlation with inhibition of p53 upregulation and mitochondrial damage.

Apoptosis plays a crucial role in clonal deletion in the thymus, and NO has been shown to prevent apoptosis in some cell types. Therefore, we examined the effect of NO on gamma-irradiation-induced thymocyte apoptosis. Treatment of 5 Gy gamma-irradiated thymocytes with 1 mM SNAP reduced cell death from 78 to 49% after 8 h incubation (spontaneous cell death in medium control cells was 26%). Coincubation with ZVAD blocked both the spontaneous cell death and the cell death induced by SNAP or gamma-irradiation. The gamma-irradiation-induced increase in caspase 3 and 6 activities was inhibited in the presence of SNAP. The increase in cytosolic cytochrome c as well as the decrease in mitochondrial membrane potential after gamma-irradiation was inhibited in the presence of SNAP. SNAP treatment also decreased the p53 upregulation in gamma-irradiated cells. In summary, we found that NO exerts a protective effect on mouse thymocyte apoptosis induced by gamma-irradiation. The mechanism of this protective effect may involve inhibition of p53 upregulation and reduction in mitochondrial damage, with subsequent inhibition of downstream caspase activation.

Outcome after steroid withdrawal in pediatric renal transplant patients receiving tacrolimus-based immunosuppression.

BACKGROUND: Corticosteroids have always been an integral part of immunosuppressive regimens in renal transplantation. The primary goal of this analysis was to assess the safety of steroid withdrawal in our pediatric renal transplant recipients receiving tacrolimus-based immunosuppression. METHODS: Between December 1989 and December 1996, 82 renal transplantations were performed in pediatric patients receiving tacrolimus-based immunosuppression. Two of these patients lost their grafts within 3 weeks of transplantation (and were still on steroids at the time of graft loss), and were excluded from further analysis. Seventy-four patients (92.5%) were taken off prednisone a median of 5.7 months after transplantation. Of these 74, 56 (70%) remained off prednisone (OFF), and 18 (22.5%) were restarted on prednisone a median of 14.8 months after discontinuing steroids (OFF --> ON). 6(7.5%) were never taken off prednisone (ON). The mean follow-up was 59 +/- 23 months. RESULTS: The 1-, 3-, and 5-year actuarial patient survival rates in the OFF group were 100%, 98%, and 96%, respectively; in the OFF --> ON group, they were 100%, 100%, and 100%, and in the ON group, they were 100%, 83%, and 83%. The 1-, 3-, and 5- year actuarial graft survival rates in the OFF group were 100%, 95%, and 82%, respectively; in the OFF --> ON group, they were 100%, 89%, and 83%; and in the ON group, they were 100%, 50%, and 33%. Two of the six graft losses in the OFF group, three out of four in the OFF --> ON Group, and two out of five in the ON group, were to chronic rejection. A time-dependent Cox regression analysis showed that the hazard for graft failure for those who came and stayed off prednisone was 0.178 relative to those who were never withdrawn from prednisone (P=0.005). Patients who were 10 years of age or younger were withdrawn from prednisone earlier (median: 5 months) than those older than 10 years (median: 7.3 months, P=0.02). In addition, patients who never had acute rejection were withdrawn from steroids earlier (median: 5 months) than those who had one or more episodes of acute rejection (median: 7.6 months, P=0.001). There was no effect of donor age, race, sex, recipient race, sex, cadaveric versus living donor, 48-hr graft function, panel reactive antibody, and total HLA mismatches or matches on the likelihood of being weaned off steroids. Serum creatinine at most recent follow-up in the OFF group was 1.2 +/- 0.5 mg/dl; in the OFF --> ON group, it was 1.8 +/- 0.9 mg/dl, and in the ON group it was 2.0 mg/dl (P<0.003). The incidence of rejection in the OFF, OFF --> ON, and ON groups was 39%, 77%, and 100%, respectively (P<0.05). CONCLUSION: These data suggest that steroid withdrawal in pediatric renal transplant patients receiving tacrolimus-based immunosuppression is associated with reasonable short- and medium-term patient and graft survival, and acceptable renal function. Patients who discontinue and then resume steroids had patient and graft survival rates comparable with those in patients who discontinue and stay off steroids, but had a higher serum creatinine and a higher incidence of rejection.

Nitric oxide induces thymocyte apoptosis via a caspase-1-dependent mechanism.

We previously showed that NO induces apoptosis in thymocytes via a p53-dependent pathway. In the present study, we investigated the role of caspases in this process. The pan-caspase inhibitor, ZVAD-fmk, and the caspase-1 inhibitor, Ac-YVAD-cho, both inhibited NO-induced thymocyte apoptosis in a dose-dependent manner, whereas the caspase-3 inhibitor, Ac-DEVD-cho, had little effect even at concentrations up to 500 microM. ZVAD-fmk and Ac-YVAD-cho were able to inhibit apoptosis when added up to 12 h, but not 16 h, after treatment with the NO donor S-nitroso-N-acetyl penicillamine (SNAP). Caspase-1 activity was up-regulated at 4 h and 8 h and returned to baseline by 24 h; caspase-3 activity was not detected. Cytosolic fractions from SNAP-treated thymocytes cleaved the inhibitor of caspase-activated deoxyribonuclease. Such cleavage was completely blocked by Ac-YVAD-cho, but not by Ac-DEVD-cho or DEVD-fmk. Poly(ADP-ribose) polymerase (PARP) was also cleaved in thymocytes 8 h and 12 h after SNAP treatment; addition of Ac-YVAD-cho to the cultures blocked PARP cleavage. Furthermore, SNAP induced apoptosis in 44% of thymocytes from wild-type mice; thymocytes from caspase-1 knockout mice were more resistant to NO-induced apoptosis. These data suggest that NO induces apoptosis in thymocytes via a caspase-1-dependent but not caspase-3-dependent pathway. Caspase-1 alone can cleave inhibitor of caspase-activated deoxyribonuclease and lead to DNA fragmentation, thus providing a novel pathway for NO-induced thymocyte apoptosis.

Inducible nitric oxide synthase (iNOS) expression upregulates p21 and inhibits vascular smooth muscle cell proliferation through p42/44 mitogen-activated protein kinase activation and independent of p53 and cyclic guanosine monophosphate.

OBJECTIVE: Overexpression of the inducible nitric oxide synthase (iNOS) gene inhibits neointimal hyperplasia after arterial injury. The purpose of this study was to examine the mechanism by which nitric oxide (NO) inhibits vascular smooth muscle cell (VSMC) proliferation, specifically focusing on signaling pathways known to be activated by NO, including cyclic guanosine monophosphate (cGMP), p53, and p42/44 mitogen-activated protein kinase (MAPK). METHODS AND RESULTS: VSMCs that were subjected to iNOS gene transfer demonstrated a reduction in proliferation (80%) that was associated with a marked increase in p21 expression. The antiproliferative and p21 stimulatory effects of NO were not suppressed by the soluble guanylate cyclase inhibitor ODQ, implicating cGMP-independent signaling. The role of p53 in NO-mediated upregulation of p21 and inhibition of proliferation was evaluated using p53 -/- VSMCs. A similar reduction in cellular proliferation and upregulation of p21 expression were achieved with iNOS gene transfer as well as treatment with the NO-donor S-nitroso-N-acetylpenicillamine (SNAP), demonstrating the p53-independent nature of these NO-mediated pathways. The transfer of the iNOS gene activated the p42/44 MAPK, and inhibition of this MAPK pathway with PD98059 partially blocked the antiproliferative effects of NO and completely inhibited the p21 stimulatory effects of NO. For confirmation that iNOS overexpression upregulated p21 in vivo, injured rat carotid arteries were infected with an adenoviral vector carrying the iNOS gene and demonstrated a marked upregulation of p21 expression at three days. However, the ability of NO to inhibit VSMC proliferation does not solely depend on p21 upregulation since the NO-donor SNAP-inhibited VSMC proliferation in p21 -/- VSMCs. CONCLUSION: Nitric oxide inhibits VSMC proliferation in association with the upregulation of p21; both occur independent of p53 and cGMP while being partially mediated through the p42/44 MAPK signaling cascade. This represents one potential mechanism by which NO inhibits VSMC proliferation.

Endothelial nitric oxide synthase protects aortic allografts from the development of transplant arteriosclerosis.

BACKGROUND: Inducible nitric oxide synthase (iNOS) is up-regulated in rejecting allografts and is protective against allograft arteriosclerosis; it suppresses neointimal smooth muscle cell accumulation and inhibits adhesion of platelets and leukocytes to the endothelium. However, the functional importance of endothelial NOS (eNOS) in the rejecting allografts remains unclear. METHODS: We examined the effects of selective eNOS deficiency in aortic allografts in a murine chronic rejection model using grafts from eNOS knockout (KO) mice (C57BL/6 background; H2b) and normal C3H (H2K) as recipients. Grafts from wild-type C57BL/6 mice served as controls. Grafts from iNOS KO mice served as a second group of controls where the contribution from iNOS was eliminated but eNOS was preserved. Aortic grafts were harvested and analyzed at days 10-14, 18-22, and 26-30 after transplantation. RESULTS: Endothelial NOS-deficient grafts showed significantly increased intima/media ratios at days 26-30 compared to controls. Immunostaining demonstrated that in eNOS KO grafts, eNOS was not detectable whereas iNOS was expressed prominently in infiltrating recipient mononuclear cells. In control grafts, eNOS expression was preserved in the endothelium even by day 30, and associated with a decrease in intimal thickening. We further demonstrated that early overexpression of iNOS by ex vivo gene transfer completely prevented the development of arteriosclerosis associated with eNOS deficiency. CONCLUSIONS: We found that eNOS plays a protective role in allografts, and that in eNOS-deficient allografts, early overexpression of iNOS is capable of preventing the development of allograft arteriosclerosis. In allografts with dysfunctional vascular endothelium and impaired eNOS activity as a result of ischemia or native arteriosclerotic disease, iNOS gene therapy may serve to improve their long-term survival and function.