Impact of heterozygous ALK1 mutations on the transcriptomic response to BMP9 and BMP10 in endothelial cells from hereditary hemorrhagic telangiectasia and pulmonary arterial hypertension donors.

Heterozygous activin receptor-like kinase 1 (ALK1) mutations are associated with two vascular diseases: hereditary hemorrhagic telangiectasia (HHT) and more rarely pulmonary arterial hypertension (PAH). Here, we aimed to understand the impact of ALK1 mutations on BMP9 and BMP10 transcriptomic responses in endothelial cells. Endothelial colony-forming cells (ECFCs) and microvascular endothelial cells (HMVECs) carrying loss of function ALK1 mutations were isolated from newborn HHT and adult PAH donors, respectively. RNA-sequencing was performed on each type of cells compared to controls following an 18 h stimulation with BMP9 or BMP10. In control ECFCs, BMP9 and BMP10 stimulations induced similar transcriptomic responses with around 800 differentially expressed genes (DEGs). ALK1-mutated ECFCs unexpectedly revealed highly similar transcriptomic profiles to controls, both at the baseline and upon stimulation, and normal activation of Smad1/5 that could not be explained by a compensation in cell-surface ALK1 level. Conversely, PAH HMVECs revealed strong transcriptional dysregulations compared to controls with > 1200 DEGs at the baseline. Consequently, because our study involved two variables, ALK1 genotype and BMP stimulation, we performed two-factor differential expression analysis and identified 44 BMP9-dysregulated genes in mutated HMVECs, but none in ECFCs. Yet, the impaired regulation of at least one hit, namely lunatic fringe (LFNG), was validated by RT-qPCR in three different ALK1-mutated endothelial models. In conclusion, ALK1 heterozygosity only modified the BMP9/BMP10 regulation of few genes, including LFNG involved in NOTCH signaling. Future studies will uncover whether dysregulations in such hits are enough to promote HHT/PAH pathogenesis, making them potential therapeutic targets, or if second hits are necessary.

Evolución en el corto plazo del compromiso renal en niños con enfermedad por el coronavirus 2019 / Course of renal involvement in the short term in children with coronavirus disease 2019

El compromiso renal en los pacientes pediátricos con enfermedad por el coronavirus 2019 (COVID-19, por su sigla en inglés) varía entre el 10 % y el 80 %. Dado que existe limitada información sobre su pronóstico, se realizó este estudio con el objetivo de describir la evolución en el corto plazo de pacientes a quienes se les detectó compromiso renal durante la internación por COVID-19. Estudio observacional y transversal que incluyó pacientes entre 1 mes y 18 años con COVID-19 con compromiso renal. Se excluyeron aquellos con patología renal conocida. Se identificaron 27 pacientes con afectación renal, en 14 de ellos se pudo realizar seguimiento para estudiar la evolución renal luego de 3 meses del diagnóstico. Todos habían normalizado los niveles de creatinina plasmática durante la internación y al momento del control ambulatorio, realizado a los 145 días (92-193), todos se encontraban normotensos y con hallazgos urinarios normales, excepto uno que persistía con microhematuria. La evolución fue favorable; la mayoría de los pacientes presentaron remisión completa del compromiso renal.

Renal involvement among pediatric patients with coronavirus disease 2019 (COVID-19) ranges between 10 % and 80 %.Given the limited information about its prognosis, the objective of this study was to describe the short-term course of patients in whom renal involvement was detected during hospitalization due to COVID-19. This was an observational, cross-sectional study in patients aged 1 month to 18 years who had COVID-19 and renal involvement. Those with a known kidney disease were excluded. A total of 27 patients with renal involvement were identified; 14 of them were followed-up to study their disease course for 3 months after diagnosis. All of the patients had achieved normal plasma creatinine levels during hospitalization and, at the time of outpatient follow-up, which took place 145 days (92-193) later, all had normal blood pressure and urinary values, except for 1 patient who continued with microscopic hematuria. Course was favorable; in most patients, renal involvement had fully resolved.

The hemodynamic and metabolic profiles of Zucker diabetic fatty rats treated with a single molecule triple vasopeptidase inhibitor, CGS 35601.

CGS 35601 is a triple vasopeptidase inhibitor (VPI) of angiotensin converting enzyme (ACE), neutral endopeptidase (NEP), and endothelin (ET) converting enzyme-1 (ECE-1), with respective IC(50) values of 22, 2, and 55 nM. The aim of the present study was to establish the hemodynamic profile of Zucker diabetic fatty (Zdf)-Fatty rats, a high-fat diet gene-prone model developing spontaneous Type 2 diabetes (T2D) and the effects of CGS 35601. Male Zdf-Fatty (14 weeks, n = 17-23), Zdf-Lean (14 weeks, n = 8-10), and Wistar (14 weeks, n = 9-10) rats on distinct diets were implanted with a catheter in the left carotid and placed individually in a metabolic cage for 30 days. The hemodynamic profile and some metabolic biomarkers were assessed daily. After a 7-day stabilization period, the Zdf-Fatty rats were divided into two groups: Group 1, controls (n = 7-10) receiving vehicle-saline (250 microl/hr) and Group 2, (n = 10-13) receiving increasing doses of CGS 35601 (0.1, 1, and 5 mg/kg/day x 6 days each, intra-arterially) followed by a 5-day washout period. Mean arterial blood pressure (MABP) of young Zdf-Fatty rats was compared with age-matched Zdf-Lean and Wistar rats, which were found similar. MABP decreased by 5.9% (from baseline at 102 +/- 5 to 96 +/- 4 mmHg), 12.7% (to 89 +/- 6 mmHg) and 21.6% (to 80 +/- 4 mmHg), at 0.1, 1, and 5 mg/kg/day, respectively, in CGS 35601-treated Zdf-Fatty rats. Systolic and diastolic blood pressures were similarly reduced. The heart rate was not affected. Hyperglycemic status and insulin-resistance were not modulated by short-term treatment. CGS 35601 presented an excellent short-term safety profile. This novel molecule and class of VPI may be of interest for lowering vascular tone. Further long-term studies, once cardiovascular and renal complications have developed in this T2D rat model are warranted to define the efficacy of this class of VPI.

Triple VPI CGS 35601 reduces high blood pressure in low-renin, high-salt Dahl salt-sensitive rats.

We previously reported that CGS 35601, a potent triple inhibitor of angiotensin-converting enzyme, neutral endopeptidase, and endothelin-converting enzyme 1, completely normalized mean arterial blood pressure (MABP) in 36-week-old spontaneously hypertensive rats, a normal renin model. The aim of the present study was to determine the effects of this triple vasopeptidase inhibitor (VPI) on the hemodynamic profile of instrumented, conscious, and unrestrained Dahl salt-sensitive (DSS) rats, a gene-prone, high-salt diet-induced low-renin hypertension model. Male DSS rats (mean weight [+/-SEM], 385 +/- 10 g) were fed a normal diet (Group 1) or a high-salt diet (Groups 2 and 3; 8% NaCl in food) for 6 weeks and then instrumented with a carotid catheter and placed individually in metabolic cages for 30 days. The hemodynamic, hematological, and biochemical profiles were assessed daily. Dose-dependent treatment started after a 7-day stabilization period in Groups 1 and 2 (vehicle dosage, 250 microl/hr) and Group 3 (CGS 35601 dosages of 0.1, 1, and 5 mg/kg/day for 6 days per dose by means of constant intra-arterial infusion), followed by a 5-day washout period. Two additional groups included normotensive Wistar rats (Group 4) and DSS rats that received a double high-salt solid (8% NaCl) and liquid (1% NaCl) diet (Group 5). The MABP in rats receiving CGS 35601 decreased in a dose-dependent fashion toward the baseline level observed in DSS rats receiving a normal diet. The heart rate was unaffected. The hemodynamic profile returned to normal during the washout period. This novel triple VPI is a potent and effective antihypertensive agent with a safe short-term profile that may be of interest for treating hypertension and other cardiovascular diseases. Other hypertensive rat models are being tested.

The first preclinical pharmacotoxicological safety assessment of CGS 35601, a triple vasopeptidase inhibitor, in chronically instrumented, conscious, and unrestrained spontaneously hypertensive rats.

CGS 35601 is a triple vasopeptidase inhibitor (VPI) of angiotensin-converting enzyme, neutral endopeptidase, and endothelin-converting enzyme-1 with respective IC50 values of 22, 2, and 55 nM. We characterized the safety profile and toxicity of escalating doses of CGS 35601 over a 20-day period in chronically instrumented, unrestrained, conscious, male, spontaneously hypertensive rats (SHR). Once instrumented with an arterial catheter, the SHR were placed in metabolic cages allowing daily assessment of hemodynamics and blood sampling for biochemical and hematological measurements. After a 7-day stabilization period, the SHR were divided into 2 groups: Gr. 1, (n = 13 to 18) receiving CGS 35601 at 0.01, 0.1, 1 and 5 mg kg(-1) day(-1) (continuous i.a. infusion) for 5 consecutive days/dose, followed by a 5-day washout; and Gr. 2, (n = 10) receiving vehicle (saline). The highest dose of CGS 35601 dose-dependently reduced MABP from 156 +/- 4 up to 94 +/- 5 mm Hg, whereas heart rate, metabolic, electrolytic, and hematological profiles, growth, diuresis, and renal activity were unaffected, and no hepatic or liver toxicities were observed. These results suggest that this novel triple VPI presents no safety concerns at this stage and may become of interest for the treatment of hypertension and other cardiovascular disorders. Long-term chronic experiments are needed to assess possible angioedema and increases in vascular permeability.

Deregulation of cyclooxygenase and nitric oxide synthase gene expression in the inflammatory cascade triggered by experimental group B streptococcal meningitis in the newborn brain and cerebral microvessels.

Group B Streptococcus (GBS) is the most common cause of neonatal sepsis and meningitis. Despite antibiotics, GBS in the newborn initiates a cascade of molecular and biological events leading to altered cerebral perfusion, blood-brain barrier disruption, cerebral edema, intracranial hypertension, neurological damage, and even death. Having previously shown that GBS infection impairs cerebral blood flow autoregulation and increases prostaglandin (PG) levels, we examined the regulation of some crucial inflammatory mediators (PGs, nitric oxide (NO), tumor necrosis factor-a) in the brain and cerebral microvessels (MVs) from newborn piglets. Cyclooxygenase (COX), the key enzyme in PG biosynthesis, exists in two isoforms, COX-1 and COX-2. Both may be directly induced by NO in a model of renal inflammation. Besides its neurotransmitter role, NO is a potent vasorelaxant whose production is catalyzed by at least three distinct nitric oxide synthases (NOS) (bNOS, ecNOS, iNOS). Western blot analyses showed that the newborn (4 day old) brain expressed lower levels of COX-1 (8-fold), COX-2 (20-fold), bNOS (12-fold), and ecNOS (5-fold) than in the 1 day old. MV showed approximately equal levels of COX-2, lower levels of COX-1 (4-fold), bNOS (5-fold), and higher levels of ecNOS (20-fold) in comparison to 4-day-old cerebral MV. A 4-day-old brain expressed lower levels of bNOS (5-fold), ecNOS (10-fold), and COX-1 (2-fold) than the 6-week-old pig. COX-2 protein was undetected in a 4-day-old pig brain, but present in great excess in MV. Purified MV showed lower ecNOS (14-fold), COX-1 (2-fold), and about equal levels of bNOS and COX-2 in comparison with MV from 6-week-old pigs. Reverse transcription polymerase chain reaction analyses confirmed these results. Treatment with noo-nitro-L-arginine (LNA), a NOS inhibitor, downregulated COX-1 expression in the newborn brain and both COX-1 and COX-2 cerebral MV expression. GBS infection (10(9) colony-forming units, 0.5 mL intracerebroventricular) of sedated newborn piglets induced the expression of tumor necrosis factor-alpha in the cerebrospinal fluid after 2 hours, upregulated bNOS expression in both brain and MVs, upregulated ecNOS in MVs, and downregulated COX-1, COX-2, and ecNOS in the brain. GBS did not trigger the expression of iNOS. Our data suggest that there is a net deficiency of NOS isoforms in the immature brain and microvasculature of the 4-day-old piglet and that the differences in expression lead to the immature control of NO and PG production, rendering newborns particularly susceptible to neurological damage because of the undeveloped nature of their response mechanisms. Moreover, the GBS-induced cascade deregulates the gene expression of interacting inflammatory mediators and may cause a net vasoconstrictor/vasodilator imbalance, leading to cerebral hypertension and edema in the early stages of infection. Pharmacological manipulations of the inflammatory cascade could lead to novel therapeutic approaches for the treatment of GBS meningitis.

Hemodynamic ischemic stroke during carotid endarterectomy: an appraisal of risk and cerebral protection.

PURPOSE: The purpose of this study was to validate the commonly accepted indicators of risk of ischemic stroke that indicate the necessity for cerebral protection during carotid endarterectomy (CEA), and to examine the efficacy of high-dose thiopentone sodium (thiopental) as a cerebral protection method in patients who are at high risk of intraoperative ischemic stroke. METHOD: In a prospective study of 37 CEAs performed for symptomatic stenosis > 70%, functional and clinical indicators of risk of ischemic stroke during carotid cross-clamping were identified. Functional indicators of risk were the development of ischemic electro-encephalogram (EEG) changes and stump pressure < 25 mm Hg. Clinical indicators of risk were previous ischemic hemispheric stroke and severe bilateral disease. These indicators were correlated in all patients, some of whom had two or three coexisting indicators of risk. The EEG and stump pressure were monitored continuously during carotid occlusion in all operations. Carotid occlusion times were recorded. Intraluminal shunting was eliminated in favor of high-dose thiopental cerebral protection in all patients. Neurologic outcome was deemed to measure the efficacy of thiopental protection in patients who are identified to be at risk and, hence, in need of cerebral protection. The validity of the indicators used to identify risk of ischemic stroke during CEA was assessed. RESULTS: The absolute stroke risk was found to be 29.7% for the whole group (37 patients) and 57.9% in 19 patients who had commonly accepted indications for protective shunting. The correlation of ischemic EEG changes with stump pressure < 25 mm Hg was only 27.3%, whereas the expected correlation based on well-documented reports in the literature was 100%. The lack of correlation may have been related to the prevention of ischemic EEG changes by thiopental. There were no neurologic deficits in the series. CONCLUSIONS: The absence of neurologic deficit in the study indicated that thiopental protection was effective in preventing ischemic stroke in high-risk patients and safely replaced intraluminal shunting.

Effects of kainic acid-induced seizures and ischemia on c-fos-like proteins in rat brain.

We have analyzed the brain pattern and time-course of c-fos-like proteins expression in kainic acid-induced seizures in the rat. C-fos-like immunoreactivity increased initially in the hippocampus, notably in the dentate gyrus, at the time of the first limbic motor seizure (90 min after kainate). C-fos-like labelling progressively involved different structures of the limbic system when the rats manifested a permanent epileptic state (3-6 h). The labelling was still conspicuous 12 h after kainate treatment and progressively declined to reach control levels 48 h after kainate. This time-course is similar to that produced by kainic acid on 2-deoxyglucose consumption and correlates with the electrographic changes previously described, supporting the idea that c-fos-like immunostaining may provide a useful marker of neuronal activity, with a cellular resolution. Since anoxic-ischemic treatment produces a very slight and transient increase in c-fos-like immunostaining restricted to the fascia dentata, c-fos-like expression is seizure-related and not due to a local hypoxia or ischemia.

[Predictive value of preoperative acidity tests in ulcer recurrence after supraselective vagotomy]. / Valeur prédictive des tests d'acidité préopératoires dans la récidive ulcéreuse après vagotomie supra-sélective.

The authors have analysed the predictive value of the basal and peak gastric acidity levels, measured preoperatively, in a series of 27 supraselective vagotomies. They recommend care in the choice of this operation for hypersecretors and they propose criteria for the preoperative selection of the candidates. They suggest determination of the preoperative basal gastric acidity level and a cimetidine suppression test to identify patients at high risk for recurrence of the ulcer.