Intravital Microscopy Evidence That Methylene Blue Should Be a Vasopressor-Sparing Agent in Sepsis Vasoplegia.

Microvasculature failure is expected in sepsis and at higher amine concentrations. Therefore, special attention focused individually on microcirculation is needed. Here, we present that methylene blue can prevent leukocytes from adhering to the endothelium in a rat model of lipopolysaccharide-induced endotoxemia. As hypothesis evidence, an intravital microscopy image is presented.

Alpha 1-acid glycoprotein is upregulated in severe COVID-19 patients and decreases neutrophil NETs in SARS-CoV-2 infection.

Orosomucoid, or alpha-1 acid glycoprotein (AGP), is a major acute-phase protein expressed in response to systemic injury and inflammation. AGP has been described as an inhibitor of neutrophil migration on sepsis, particularly its immunomodulation effects. AGP's biological functions in coronavirus disease 2019 (COVID-19) are not understood. We sought to investigate the role of AGP in severe COVID-19 infection patients and neutrophils infected with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Epidemiological data, AGP levels, and other laboratory parameters were measured in blood samples from 56 subjects hospitalized in the ICU with SARS-CoV-2 infection. To evaluate the role of AGP in NETosis in neutrophils, blood samples from health patients were collected, and neutrophils were separated and infected with SARS-CoV-2. Those neutrophils were treated with AGP or vehicle, and NETosis was analyzed by flow cytometry. AGP was upregulated in severe COVID-19 patients (p<0.05). AGP level was positively correlated with IL-6 and C-reactive protein (respectively, p=0.005, p=0.002) and negatively correlated with lactate (p=0.004). AGP treatment downregulated early and late NETosis (respectively, 35.7% and 43.5%) in neutrophils infected with SARS-CoV-2 and up-regulated IL-6 supernatant culture expression (p<0.0001). Our data showed increased AGP in COVID-19 infection and contributed to NETosis regulation and increased IL-6 production, possibly related to the Cytokine storm in COVID-19.

Intravital Microscopy Evidence That Methylene Blue Should Be a Vasopressor-Sparing Agent in Sepsis Vasoplegia

ABSTRACT Microvasculature failure is expected in sepsis and at higher amine concentrations. Therefore, special attention focused individually on microcirculation is needed. Here, we present that methylene blue can prevent leukocytes from adhering to the endothelium in a rat model of lipopolysaccharide-induced endotoxemia. As hypothesis evidence, an intravital microscopy image is presented.

Cytokine storm in individuals with severe COVID-19 decreases endothelial cell antioxidant defense via downregulation of the Nrf2 transcriptional factor.

The cytokine storm in SARS-CoV-2 infection contributes to the onset of inflammation and target-organ damage. The endothelium is a key player in COVID-19 pathophysiology and it is an important target for cytokines. Considering that cytokines trigger oxidative stress and negatively impact endothelial cell function, we sought to determine whether serum from individuals with severe COVID-19 decreases endothelial cells' main antioxidant defense, i.e., the antioxidant transcriptional factor Nrf2. Human umbilical vein endothelial cells (HUVECs) were incubated with serum from patients with severe COVID-19 at different time points and the effects on redox balance and Nrf2 activity were determined. Serum from individuals with COVID-19 increased oxidant species, as indicated by higher DHE (dihydroethydine) oxidation, increased protein carbonylation, and induced mitochondrial reactive oxygen species (ROS) generation and dysfunction. Serum from patients with COVID-19, but not serum from healthy individuals, induced cell death and diminished nitric oxide (NO) bioavailability. In parallel, Nrf2 nuclear accumulation and the expression of Nrf2-targeted genes were decreased in endothelial cells exposed to serum from individuals with COVID-19. In addition, these cells exhibited higher expression of Bach-1, a negative regulator of Nrf2 that competes for DNA binding. All events were prevented by tocilizumab, an IL-6 receptor blocker, indicating that IL-6 is key to the impairment of endothelial antioxidant defense. In conclusion, endothelial dysfunction related to SARS-CoV-2 infection is linked to decreased endothelial antioxidant defense via IL-6-dependent mechanisms. Pharmacological activation of Nrf2 may decrease endothelial cell damage in individuals with severe COVID-19.NEW & NOTEWORTHY We demonstrate that endothelial cell dysfunction in SARS-CoV-2-infected individuals is linked to decreased activity of the major antioxidant system regulator, the Nrf2 transcription factor. We provide evidence that this phenomenon relies on IL-6, an important cytokine involved in the pathophysiology of COVID-19. Our data support the view that Nrf2 activation is a potential therapeutical strategy to prevent oxidative stress and vascular inflammation in severe cases of COVID-19.

Inhibition of IL-6 signaling prevents serum-induced umbilical cord artery dysfunction from patients with severe COVID-19.

Coronavirus disease 2019 (COVID-19) infection has a negative impact on the cytokine profile of pregnant women. Increased levels of proinflammatory cytokines seem to be correlated with the severity of the disease, in addition to predisposing to miscarriage or premature birth. Proinflammatory cytokines increase the generation of reactive oxygen species (ROS). It is unclear how interleukin-6 (IL-6) found in the circulation of patients with severe COVID-19 might affect gestational health, particularly concerning umbilical cord function. This study tested the hypothesis that IL-6 present in the circulation of women with severe COVID-19 causes umbilical cord artery dysfunction by increasing ROS generation and activating redox-sensitive proteins. Umbilical cord arteries were incubated with serum from healthy women and women with severe COVID-19. Vascular function was assessed using concentration-effect curves to serotonin in the presence or absence of pharmacological agents, such as tocilizumab (antibody against the IL-6 receptor), tiron (ROS scavenger), ML171 (Nox1 inhibitor), and Y27632 (Rho kinase inhibitor). ROS generation was assessed by the dihydroethidine probe and Rho kinase activity by an enzymatic assay. Umbilical arteries exposed to serum from women with severe COVID-19 were hyperreactive to serotonin. This effect was abolished in the presence of tocilizumab, tiron, ML171, and Y27632. In addition, serum from women with severe COVID-19 increased Nox1-dependent ROS generation and Rho kinase activity. Increased Rho kinase activity was abolished by tocilizumab and tiron. Serum cytokines in women with severe COVID-19 promote umbilical artery dysfunction. IL-6 is key to Nox-linked vascular oxidative stress and activation of the Rho kinase pathway.

Indigo Carmine Hemodynamic Studies to Treat Vasoplegia Induced by Compound 48/80 in a Swine Model of Anaphylaxis.

INTRODUCTION: There are many reasons to believe that the nitric oxide/guanosine 3'5' - cyclic monophosphate (or NO/cGMP) pathway on vasoplegic states is underestimated. To study indigo carmine (IC) as an alternative to methylene blue was the investigation rationale. METHODS: The IC (3mg/kg intravenous infusion) study protocol included five experimental groups; 1) Control group - saline was injected at 0 and 10 minutes; 2) IC group - IC was injected at 0 and saline at 10 minutes; 3) compound 48/80 (C48/80) group - C48/80 was injected at 0 minute and saline at 10 minutes; 4) C48/80 + IC group - C48/80 was injected at 0 minute and IC at 10 minutes; and 5) IC + C48/80 group - IC was injected at 0 minute and C48/80 at 10 minutes. The studies were carried out by registering and measuring hemodynamic and blood gasometric parameters, including continuous cardiac output. RESULTS: 1) The effects of the drugs (IC and C48/80) were more evident in the first 20 minutes of recording; 2) hypotensive responses were more pronounced in the C48/80 groups; 3) IC isolated or applied before C48/80 caused transient pulmonary hypertension; and 4) after the first 20 minutes, the pressure responses showed stability with apparent hypotension more pronounced in the C48/80 groups. Clinical observations showed significant hemodynamic instability and catastrophic anaphylactic reactions (agitation, pulmonary hypertension, severe bronchospasm, urticaria, high-intensity cyanosis, violent gastric hypersecretion, and ascites). CONCLUSION: A global results analysis showed differences between groups only in the first 20 minutes of the experiments.

Indigo Carmine Hemodynamic Studies to Treat Vasoplegia Induced by Compound 48/80 in a Swine Model of Anaphylaxis

Abstract Introduction: There are many reasons to believe that the nitric oxide/guanosine 3'5' - cyclic monophosphate (or NO/cGMP) pathway on vasoplegic states is underestimated. To study indigo carmine (IC) as an alternative to methylene blue was the investigation rationale. Methods: The IC (3mg/kg intravenous infusion) study protocol included five experimental groups; 1) Control group — saline was injected at 0 and 10 minutes; 2) IC group — IC was injected at 0 and saline at 10 minutes; 3) compound 48/80 (C48/80) group — C48/80 was injected at 0 minute and saline at 10 minutes; 4) C48/80 + IC group — C48/80 was injected at 0 minute and IC at 10 minutes; and 5) IC + C48/80 group — IC was injected at 0 minute and C48/80 at 10 minutes. The studies were carried out by registering and measuring hemodynamic and blood gasometric parameters, including continuous cardiac output. Results: 1) The effects of the drugs (IC and C48/80) were more evident in the first 20 minutes of recording; 2) hypotensive responses were more pronounced in the C48/80 groups; 3) IC isolated or applied before C48/80 caused transient pulmonary hypertension; and 4) after the first 20 minutes, the pressure responses showed stability with apparent hypotension more pronounced in the C48/80 groups. Clinical observations showed significant hemodynamic instability and catastrophic anaphylactic reactions (agitation, pulmonary hypertension, severe bronchospasm, urticaria, high-intensity cyanosis, violent gastric hypersecretion, and ascites). Conclusion: A global results analysis showed differences between groups only in the first 20 minutes of the experiments.

Heparin prevents in vitro glycocalyx shedding induced by plasma from COVID-19 patients.

The severe forms and worsened outcomes of COVID-19 (coronavirus disease 19) are closely associated with hypertension and cardiovascular disease. Endothelial cells express Angiotensin-Converting Enzyme 2 (ACE2), which is the entrance door for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The hallmarks of severe illness caused by SARS-CoV-2 infection are increased levels of IL-6, C-reactive protein, D-dimer, ferritin, neutrophilia and lymphopenia, pulmonary intravascular coagulopathy and microthrombi of alveolar capillaries. The endothelial glycocalyx, a proteoglycan- and glycoprotein-rich layer covering the luminal side of endothelial cells, contributes to vascular homeostasis. It regulates vascular tonus and permeability, prevents thrombosis, and modulates leukocyte adhesion and inflammatory response. We hypothesized that cytokine production and reactive oxygen species (ROS) generation associated with COVID-19 leads to glycocalyx degradation. A cohort of 20 hospitalized patients with a confirmed COVID-19 diagnosis and healthy subjects were enrolled in this study. Mechanisms associated with glycocalyx degradation in COVID-19 were investigated. Increased plasma concentrations of IL-6 and IL1-ß, as well as increased lipid peroxidation and glycocalyx components were detected in plasma from COVID-19 patients compared to plasma from healthy subjects. Plasma from COVID-19 patients induced glycocalyx shedding in cultured human umbilical vein endothelial cells (HUVECs) and disrupted redox balance. Treatment of HUVECs with low molecular weight heparin inhibited the glycocalyx perturbation. In conclusion, plasma from COVID-19 patients promotes glycocalyx shedding and redox imbalance in endothelial cells, and heparin treatment potentially inhibits glycocalyx disruption.

Chronic Intermittent Hypoxia Triggers a Senescence-like Phenotype in Human White Preadipocytes.

Obstructive sleep apnea (OSA) is a common sleep disorder associated with obesity. Emerging evidence suggest that OSA increases the risk of cardiovascular morbidity and mortality partly via accelerating the process of cellular aging. Thus, we sought to examine the effects of intermittent hypoxia (IH), a hallmark of OSA, on senescence in human white preadipocytes. We demonstrate that chronic IH is associated with an increased generation of mitochondrial reactive oxygen species along with increased prevalence of cells with nuclear localization of γH2AX & p16. A higher prevalence of cells positive for senescence-associated ß-galactosidase activity was also evident with chronic IH exposure. Intervention with aspirin, atorvastatin or renin-angiotensin system (RAS) inhibitors effectively attenuated IH-mediated senescence-like phenotype. Importantly, the validity of in vitro findings was confirmed by examination of the subcutaneous abdominal adipose tissue which showed that OSA patients had a significantly higher percentage of cells with nuclear localization of γH2AX & p16 than non-OSA individuals (20.1 ± 10.8% vs. 10.3 ± 2.7%, Padjusted < 0.001). Furthermore, the frequency of dual positive γH2AX & p16 nuclei in adipose tissue of OSA patients receiving statin, aspirin, and/or RAS inhibitors was comparable to non-OSA individuals. This study identifies chronic IH as a trigger of senescence-like phenotype in preadipocytes. Together, our data suggest that OSA may be considered as a senescence-related disorder.

Telomere Length and Risk of Major Adverse Cardiac Events and Cancer in Obstructive Sleep Apnea Patients.

Telomere length (TL) is associated with cardiovascular disease (CVD) and cancer. Obstructive sleep apnea (OSA) is also linked to higher risk of CVD and cancer, and to TL. We investigated the association between TL and risk of major adverse cardiac events (MACE) and cancer in OSA patients. We studied 210 individuals undergoing sleep-related studies between 2000 and 2007. Baseline characteristics and follow-up data (available in 164 subjects) were obtained from clinic records. Incidence rates were calculated for the entire group and by OSA status. Hazard ratios were calculated to estimate effects of OSA and TL on risk of MACE and cancer. In total, 32 individuals (20%) developed MACE and/or cancer during 12.7-year follow-up. The OSA group had a higher likelihood of cancer (16.0 vs. 4.9 events per 1000 person-years, P = 0.044) but no clear evidence of an elevated incidence of MACE (10.8 vs. 4.8 events per 1000 person-years, P = 0.293) compared to the non-OSA group. There was no association between TL and MACE- (HR = 1.01, 95% CI 0.78-1.28), or cancer-risk (HR = 1.18, 95% CI 0.96-1.43). Our study warrants further investigation of any modulating effect of OSA on TL and the risk of MACE and cancer.

Comparative Expression of Renin-Angiotensin Pathway Proteins in Visceral Versus Subcutaneous Fat.

Body fat distribution contributes to obesity-related metabolic and cardiovascular disorders. Visceral fat is more detrimental than subcutaneous fat. However, the mechanisms underlying visceral fat-mediated cardiometabolic dysregulation are not completely understood. Localized increases in expression of the renin angiotensin system (RAS) in adipose tissue (AT) may be implicated. We therefore investigated mRNA and protein expression of RAS components in visceral versus subcutaneous AT using paired samples from individuals undergoing surgery (N = 20, body mass index: 45.6 ± 6.2 kg/m2, and age: 44.6 ± 9.1 years). We also examined RAS-related proteins in AT obtained from individuals on renin angiotensin aldosterone system (RAAS) targeted drugs (N = 10, body mass index: 47.2 ± 9.3 kg/m2, and age: 53.3 ± 10.1 years). Comparison of protein expression between subcutaneous and visceral AT samples showed an increase in renin (p = 0.004) and no change in angiotensinogen (p = 0.987) expression in visceral AT. Among proteins involved in angiotensin peptide generation, angiotensin converting enzyme (p = 0.02) was increased in subcutaneous AT while chymase (p = 0.001) and angiotensin converting enzyme-2 (p = 0.001) were elevated in visceral fat. Furthermore, visceral fat expression of angiotensin II type-2 receptor (p = 0.007) and angiotensin II type-1 receptor (p = 0.031) was higher, and MAS receptor (p < 0.001) was lower. Phosphorylated-p53 (p = 0.147), AT fibrosis (p = 0.138) and average adipocyte size (p = 0.846) were similar in the two depots. Nonetheless, visceral AT showed increased mRNA expression of inflammatory (TNFα, p < 0.001; IL-6, p = 0.001) and oxidative stress markers (NOX2, p = 0.038; NOX4, p < 0.001). Of note, mRNA and protein expression of RAS components did not differ between subjects taking or not taking RAAS related drugs. In summary, several RAS related proteins are differentially expressed in subcutaneous versus visceral AT. This differential expression may not alter AngII but likely increases Ang1-7 generation in visceral fat. These potential differences in active angiotensin peptides and receptor expression in the two depots suggest that localized RAS may not be involved in differences in visceral vs subcutaneous AT function in obese individuals. Our findings do not support a role for localized RAS differences in visceral fat-mediated development of cardiovascular and metabolic pathology.

The Role of Interleukins and Inflammatory Markers in the Early Restenosis of Covered Stents in the Femoropopliteal Arterial Segment.

BACKGROUND: The objective of this study was to evaluate the relationship between inflammatory markers, such as interleukin (IL)-1ß, IL-6, IL-8, IL-10, tumor necrosis factor α (TNF-α), transforming growth factor ß (TGF-ß), and highly sensitive C-reactive protein, and the development of arterial restenosis 6 months after femoropopliteal percutaneous transluminal angioplasty (PTA) with covered stent implantation. METHODS: We recruited 27 patients of a tertiary hospital in Brazil who were treated with covered stents for atherosclerotic peripheral arterial disease. Serum samples were collected before stent implantation, then 24 hr later, and 6 months after the procedure. RESULTS: At 6-month follow-up, 4 patients (15%) presented restenosis. IL1- ß, IL-6, IL-8, and TNF-α levels showed a statistically significant reduction after both 24 hr and 6 months compared with pretreatment levels (P < 0.01). There were increased levels of IL-10 and TGF-ß both 24 hr and 6 months after PTA and stenting compared with pretreatment levels (P < 0.01). None of the cytokines studied were correlated with restenosis. CONCLUSIONS: This study demonstrated a significant increase in anti-inflammatory TGF-ß and IL-10 and a decrease in proinflammatory cytokines IL-1ß, IL-6, IL-8, and TNF-α 6 months after the procedure, but no inflammatory marker was independently identified as a risk factor for in-stent restenosis.

Carotid sinus nerve electrical stimulation in conscious rats attenuates systemic inflammation via chemoreceptor activation.

Recent studies demonstrated a critical functional connection between the autonomic (sympathetic and parasympathetic) nervous and the immune systems. The carotid sinus nerve (CSN) conveys electrical signals from the chemoreceptors of the carotid bifurcation to the central nervous system where the stimuli are processed to activate sympathetic and parasympathetic efferent signals. Here, we reported that chemoreflex activation via electrical CSN stimulation, in conscious rats, controls the innate immune response to lipopolysaccharide attenuating the plasma levels of inflammatory cytokines such as tumor necrosis factor (TNF), interleukin 1ß (IL-1ß) and interleukin 6 (IL-6). By contrast, the chemoreflex stimulation increases the plasma levels of anti-inflammatory cytokine interleukin 10 (IL-10). This chemoreflex anti-inflammatory network was abrogated by carotid chemoreceptor denervation and by pharmacological blockade of either sympathetic - propranolol - or parasympathetic - methylatropine - signals. The chemoreflex stimulation as well as the surgical and pharmacological procedures were confirmed by real-time recording of hemodynamic parameters [pulsatile arterial pressure (PAP) and heart rate (HR)]. These results reveal, in conscious animals, a novel mechanism of neuromodulation mediated by the carotid chemoreceptors and involving both the sympathetic and parasympathetic systems.

The role of the kallikrein-kinin system, matrix metalloproteinases, and tissue inhibitors of metalloproteinases in the early restenosis of covered stents in the femoropopliteal arterial segment.

OBJECTIVE: The purpose of this study was to investigate the roles of the kallikrein-kinin system and matrix metalloproteinases (MMPs) in the development of arterial restenosis attributable to intimal hyperplasia in the femoropopliteal arteries. METHODS: This report describes a single-center prospective study of 27 patients with peripheral artery disease who required percutaneous transluminal angioplasty and stenting of the femoropopliteal segment using covered stent grafts. The blood concentrations of total and kininogen fractions were evaluated using immunoenzymatic methods. Plasma kallikrein was evaluated by the colorimetric method. Tissue kallikrein was evaluated by the spectrophotometric method. The activity of kininase II was measured by fluorometric analysis. Quantification of MMPs was performed by zymography, and tissue inhibitors of metalloproteinases were measured by enzyme-linked immunosorbent assay. RESULTS: Four (15%) of the treated patients developed restenosis at the 6-month follow-up evaluation. These patients had significantly lower levels of high-molecular-weight kininogens (24 hours; P < .05) and low-molecular-weight kininogens (before, P < .05; 24 hours, P < .01; 6 months, P < .05) and lower levels of tissue inhibitor of metalloproteinases-2 (6 months; P < .05) than the patients without restenosis. The activity levels of plasma and tissue kallikrein, kininase II, and MMPs did not differ significantly between the patients with and without restenosis. CONCLUSIONS: This study demonstrates an involvement of the kallikrein-kinin system in in-stent restenosis, although we could not confirm the participation of metalloproteinases in the restenosis process.

Pyridostigmine prevents haemodynamic alterations but does not affect their nycthemeral oscillations in infarcted mice.

The increase in acetylcholine yielded by pyridostigmine (PYR), an acetylcholinesterase inhibitor, was evaluated for its effect on the haemodynamic responses-mean arterial pressure (MAP) and heart rate (HR)-and their nycthemeral oscillation in mice before and one week after myocardial infarction (MI). Mice were anesthetized (isoflurane), and a telemetry transmitter was implanted into the carotid artery. After 5 days of recovery, the MAP and HR were recorded for 48 h (10 s every 10 min). Following this procedure, mice were submitted to surgery for sham or coronary artery ligation and received drinking water (VEHICLE) with or without PYR. Five days after surgery, the haemodynamic recordings were recommenced. Sham surgery combined with VEHICLE did not affect basal MAP and HR; nevertheless, these haemodynamic parameters were higher during the night, before and after surgery. MI combined with VEHICLE displayed decreased MAP and increased HR; these haemodynamic parameters were also higher during the night, before and after surgery. Sham surgery combined with PYR displayed similar results for MAP as sham combined with VEHICLE; however, PYR produced bradycardia. Nevertheless, MI combined with PYR exhibited no change in MAP and HR, but these haemodynamic parameters were also higher during the night, before and after surgery. Therefore, MI decreased MAP and increased HR, while PYR prevented these alterations. Neither MI nor PYR affected nycthemeral oscillations of MAP and HR. These findings indicate that the increase in acetylcholine yielded by PYR protected the haemodynamic alterations caused by MI in mice, without affecting the nycthemeral haemodynamic oscillations.

Pyridostigmine restores cardiac autonomic balance after small myocardial infarction in mice.

The effect of pyridostigmine (PYR)--an acetylcholinesterase inhibitor--on hemodynamics and cardiac autonomic control, was never studied in conscious myocardial infarcted mice. Telemetry transmitters were implanted into the carotid artery under isoflurane anesthesia. Seven to ten days after recovery from the surgery, basal arterial pressure and heart rate were recorded, while parasympathetic and sympathetic tone (ΔHR) was evaluated by means of methyl atropine and propranolol. After the basal hemodynamic recording the mice were subjected to left coronary artery ligation for producing myocardial infarction (MI), or sham operation, and implantation of minipumps filled with PYR or saline. Separate groups of anesthetized (isoflurane) mice previously (4 weeks) subjected to MI, or sham coronary artery ligation, were submitted to cardiac function examination. The mice exhibited an infarct length of approximately 12%, no change in arterial pressure and increased heart rate only in the 1st week after MI. Vagal tone decreased in the 1st week, while the sympathetic tone was increased in the 1st and 4th week after MI. PYR prevented the increase in heart rate but did not affect the arterial pressure. Moreover, PYR prevented the increase in sympathetic tone throughout the 4 weeks. Concerning the parasympathetic tone, PYR not only impaired its attenuation in the 1st week, but enhanced it in the 4th week. MI decreased ejection fraction and increased diastolic and systolic volume. Therefore, the pharmacological increase of peripheral acetylcholine availability by means of PYR prevented tachycardia, increased parasympathetic and decreased sympathetic tone after MI in mice.

Characterization of the kallikrein-kinin system, metalloproteinases, and their tissue inhibitors in the in-stent restenosis after peripheral percutaneous angioplasty.

BACKGROUND: The kallikrein-kinin system (KKS) has several direct and indirect effects on cells and cellular mediators involved in the inflammatory process. Studies about inflammation on percutaneous transluminal angioplasty with stent (PTA/stent) to treat peripheral arterial disease (PAD) in humans are scarce. The matrix metalloproteinases (MMPs) are calcium-dependent zinc-containing endopeptidases expressed in various cells and tissues such as fibroblasts, inflammatory cells, and, smooth muscle cells. Changes in the extracellular matrix (ECM) take place in the pathogenesis of many cardiovascular pathologies. MMPs and their inhibitors (tissue inhibitors of metalloproteinases [TIMPs]) are crucial in ECM remodeling in both physiologic and pathologic conditions. The aim of this study was to evaluate the role of the KKS and the MMP metabolism, which are important mediators that may contribute to tissue repair, in the process of arterial restenosis due to intimal hyperplasia in the femoropopliteal segment with the aim of developing new interventions. METHODS: Thirty-nine consecutive patients were selected (regardless of ethnic group, age, or sex) for revascularization, who underwent PTA/stent of the femoropopliteal segment. Twenty-five patients with the same clinical characteristics who were scheduled for diagnostic angiography but not subjected to PTA/nitinol stent were also selected. The concentrations in blood of total and kininogen fractions were evaluated using immunoenzymatic methods. Plasma kallikrein was evaluated by the colorimetric method. Tissue kallikrein was evaluated by the spectrophotometric method. The activity of kininase II was measured by fluorometric analysis. Quantification of MMPs was performed by zymography, which is an electrophoresis technique, and TIMPs were measured by enzyme-linked immunosorbent assay. RESULTS: Among the 31 patients who completed the survey, there were 10 cases of angiographically defined restenosis of >50%, and 21 cases without restenosis. There was an increase in the concentrations of the substrates (high-molecular-weight kininogens and lower molecular weight kininogens) and enzymes (plasma and tissue kallikrein) in patients with restenosis, indicating activation of this inflammatory pathway in these patients. The activity of kininase II was not significantly different between the groups of patients studied. There were no statistical differences between restenosis and no restenosis patients for both MMPs and TIMPs dosage, but there is an upward trend of MMPs in time 6 months in patients with restenosis. CONCLUSIONS: With the aim of identifying factors contributing to restenosis after endovascular intervention, this study showed evidence of high activation of the KKS in the pathologic inflammatory process of PTA/stent restenosis. In the other hand, it could not show participation of metalloproteinase metabolism in PTA/stent restenosis.

Angiotensin II-independent angiotensin-(1-7) formation in rat hippocampus: involvement of thimet oligopeptidase.

The involvement and relevance of the renin-angiotensin system have been established clearly in cardiovascular diseases, and renin-angiotensin system involvement has also been investigated extensively in the central nervous system. Angiotensin II acts classically by binding to the AT1 and AT2 receptors. However, other pathways within the renin-angiotensin system have been described more recently, such as one in which angiotensin-(1-7) (Ang-(1-7)) binds to the receptor Mas. In the central nervous system specifically, it has been reported that this heptapeptide is involved in learning and memory processes that occur in central limbic regions, such as the hippocampus. Therefore, this prompted us to investigate the possible role of the Ang-(1-7)-receptor Mas pathway in epileptic seizures, which are also known to recruit limbic areas. In the present study, we show that Ang-(1-7) is the main metabolite of angiotensin I in rat hippocampi, and, strikingly, that thimet oligopeptidase is the main enzyme involved in the generation of Ang-(1-7). Furthermore, elevations in the levels of thimet oligopeptidase, Ang-(1-7), and of receptor Mas transcripts are observed in chronically stimulated epileptic rats, which suggest that the thimet oligopeptidase-Ang-(1-7)-receptor Mas axis may have a functional relevance in the pathophysiology of these animals. In summary, our data, which describe a new preferential biochemical pathway for the generation of Ang-(1-7) in the central nervous system and an increase in the levels of various elements of the related thimet oligopeptidase-Ang-(1-7)-receptor Mas pathway, unveil potential new roles of the renin-angiotensin system in central nervous system pathophysiology.