G-protein-coupled receptor MAS deletion produces a preeclampsia-like phenotype in FVB/N mice.

BACKGROUND: An unbalance in the renin-angiotensin (Ang) system (RAS) between the Ang II/AT1 and Ang-(1-7)/Mas axis appears to be involved in preeclampsia (PE), in which a reduction in Ang-(1-7) was observed. Here, we tested whether the reduction in the activity of the Ang-(1-7)/Mas axis could be a contributing factor for the development of PE, using Mas-deficient (Mas-/-) mice. METHODS AND RESULTS: Cardiovascular parameters were evaluated by telemetry before, during pregnancy and 4 days postpartum in 20-week-old Mas-/- and wild-type (WT) female mice. Mas-/- mice presented reduced arterial blood pressure (BP) at baseline (91.3 ± 0.8 in Mas-/- vs. 94.0 ± 0.9 mmHg in WT, Diastolic, P<0.05). However, after the 13th day of gestation, BP in Mas-/- mice started to increase, time-dependently, and at day 19 of pregnancy, these animals presented a higher BP in comparison with WT group (90.5 ± 0.7 in Mas-/- vs. 80.3 ± 3.5 mmHg in WT, Diastolic D19, P<0.0001). Moreover, pregnant Mas-/- mice presented fetal growth restriction, increase in urinary protein excretion as compared with nonpregnant Mas-/-, oliguria, increase in cytokines, endothelial dysfunction and reduced ACE, AT1R, ACE2, ET-1A, and eNOS placental mRNA, similar to some of the clinical manifestations found in the development of PE. CONCLUSIONS: These results show that Mas-deletion produces a PE-like state in FVB/N mice.

Phenylalanine hydroxylase contributes to serotonin synthesis in mice.

Serotonin is an important signaling molecule in the periphery and in the brain. The hydroxylation of tryptophan is the first and rate-limiting step of its synthesis. In most vertebrates, two enzymes have been described to catalyze this step, tryptophan hydroxylase (TPH) 1 and 2, with expression localized to peripheral and neuronal cells, respectively. However, animals lacking both TPH isoforms still exhibit about 10% of normal serotonin levels in the blood demanding an additional source of the monoamine. In this study, we provide evidence by the gain and loss of function approaches in in vitro and in vivo systems, including stable-isotope tracing in mice, that phenylalanine hydroxylase (PAH) is a third TPH in mammals. PAH contributes to serotonin levels in the blood, and may be important as a local source of serotonin in organs in which no other TPHs are expressed, such as liver and kidney.

Phosphodiesterase 3A and Arterial Hypertension.

BACKGROUND: High blood pressure is the primary risk factor for cardiovascular death worldwide. Autosomal dominant hypertension with brachydactyly clinically resembles salt-resistant essential hypertension and causes death by stroke before 50 years of age. We recently implicated the gene encoding phosphodiesterase 3A (PDE3A); however, in vivo modeling of the genetic defect and thus showing an involvement of mutant PDE3A is lacking. METHODS: We used genetic mapping, sequencing, transgenic technology, CRISPR-Cas9 gene editing, immunoblotting, and fluorescence resonance energy transfer. We identified new patients, performed extensive animal phenotyping, and explored new signaling pathways. RESULTS: We describe a novel mutation within a 15 base pair (bp) region of the PDE3A gene and define this segment as a mutational hotspot in hypertension with brachydactyly. The mutations cause an increase in enzyme activity. A CRISPR/Cas9-generated rat model, with a 9-bp deletion within the hotspot analogous to a human deletion, recapitulates hypertension with brachydactyly. In mice, mutant transgenic PDE3A overexpression in smooth muscle cells confirmed that mutant PDE3A causes hypertension. The mutant PDE3A enzymes display consistent changes in their phosphorylation and an increased interaction with the 14-3-3θ adaptor protein. This aberrant signaling is associated with an increase in vascular smooth muscle cell proliferation and changes in vessel morphology and function. CONCLUSIONS: The mutated PDE3A gene drives mechanisms that increase peripheral vascular resistance causing hypertension. We present 2 new animal models that will serve to elucidate the underlying mechanisms further. Our findings could facilitate the search for new antihypertensive treatments.

Increased angiotensin II formation in the brain modulates cardiovascular homeostasis and erythropoiesis.

In spite of the fact that the modulatory effects of angiotensin II (Ang II) on the sympathetic nerve activity to targeted organs involved in blood pressure (BP) regulation is well acknowledged, the local production of this peptide in the brain and the consequences of enhanced central Ang II beyond the cardiovascular system are not yet well comprehended. In the present study, we generated and validated a new transgenic mouse line overexpressing the rat full-length angiotensinogen (Agt) protein specifically in the brain (Agt-Tg). Adult Agt-Tg mice presented overall increased gene expression of total Agt in the brain including brainstem and hypothalamus. In addition, the excess of Agt led to abundantly detectable brain Ang II levels as well as increased circulating copeptin levels. Agt-Tg displayed raised BP in acute recordings, while long-term telemetrically measured basal BP was indistinguishable from wild-types. Agt-Tg has altered peripheral renin-angiotensin system and vasomotor sympathetic tone homeostasis because renal gene expression analysis, plasma Ang II measurements and ganglionic blockade experiments revealed suppressed renin expression and reduced Ang II and higher neurogenic pressure response, respectively. Plasma and urine screens revealed apparently normal fluid and electrolyte handling in Agt-Tg. Interestingly, hematological analyses showed increased hematocrit in Agt-Tg caused by enhanced erythropoiesis, which was reverted by submitting the transgenic mice to a long-term peripheral sympathectomy protocol. Collectively, our findings suggest that Agt-Tg is a valuable tool to study not only brain Ang II formation and its modulatory effects on cardiovascular homeostasis but also its role in erythropoiesis control via autonomic modulation.

Hemodynamic phenotyping of transgenic rats with ubiquitous expression of an angiotensin-(1-7)-producing fusion protein.

Activation of the angiotensin (Ang)-converting enzyme (ACE) 2/Ang-(1-7)/MAS receptor pathway of the renin-angiotensin system (RAS) induces protective mechanisms in different diseases. Herein, we describe the cardiovascular phenotype of a new transgenic rat line (TG7371) that expresses an Ang-(1-7)-producing fusion protein. The transgene-specific mRNA and the corresponding protein were shown to be present in all evaluated tissues of TG7371 with the highest expression in aorta and brain. Plasma Ang-(1-7) levels, measured by radioimmunoassay (RIA) were similar to control Sprague-Dawley (SD) rats, however high Ang-(1-7) levels were found in the hypothalamus. TG7371 showed lower baseline mean arterial pressure (MAP), assessed in conscious or anesthetized rats by telemetry or short-term recordings, associated with increased plasma atrial natriuretic peptide (ANP) and higher urinary sodium concentration. Moreover, evaluation of regional blood flow and hemodynamic parameters with fluorescent microspheres showed a significant increase in blood flow in different tissues (kidneys, mesentery, muscle, spleen, brown fat, heart and skin), with a resulting decrease in total peripheral resistance (TPR). TG7371 rats, on the other hand, also presented increased cardiac and global sympathetic tone, increased plasma vasopressin (AVP) levels and decreased free water clearance. Altogether, our data show that expression of an Ang-(1-7)-producing fusion protein induced a hypotensive phenotype due to widespread vasodilation and consequent fall in peripheral resistance. This phenotype was associated with an increase in ANP together with an increase in AVP and sympathetic drive, which did not fully compensate the lower blood pressure (BP). Here we present the hemodynamic impact of long-term increase in tissue expression of an Ang-(1-7)-fusion protein and provide a new tool to investigate this peptide in different pathophysiological conditions.

Angiotensin-II receptor type Ia does not contribute to cardiac atrophy following high-thoracic spinal cord injury in mice.

NEW FINDINGS: What is the central question of this study? What is the role of the renin-angiotensin system with angiotensin II acting via its receptor AT1a in spinal cord injury-induced cardiac atrophy? What is the main finding and its importance? Knockout of AT1a did not protect mice that had undergone thoracic level 4 transection from cardiac atrophy. There were no histopathological signs but there was reduced load-dependent left ventricular function (lower stroke volume and cardiac output) with preserved ejection fraction. ABSTRACT: Spinal cord injury (SCI) leads to cardiac atrophy often accompanied by functional deficits. The renin-angiotensin system (RAS) with angiotensin II (AngII) signalling via its receptor AT1a might contribute to cardiac atrophy post-SCI. We performed spinal cord transection at thoracic level T4 (T4-Tx) or sham-operation in female wild-type mice (WT, n = 27) and mice deficient in AT1a (Agtr1a-/- , n = 27). Echocardiography (0, 7, 21 and 28 days post-SCI) and histology and gene expression analyses at 1 and 2 months post-SCI were performed. We found cardiac atrophy post-SCI: reduced heart weight, reduced estimated left ventricular mass in Agtr1a-/- , and reduced cardiomyocyte diameter in WT mice. Although, the latter as well as stroke volume (SV) and cardiac output (CO) were reduced in Agtr1a-/- mice already at baseline, cardiomyocyte diameter was even smaller in injured Agtr1a-/- mice compared to injured WT mice. SV and CO were reduced in WT mice post-SCI. Ejection fraction and fractional shortening were preserved post-SCI in both genotypes. There were no histological signs of fibrosis and pathology in the cardiac sections of either genotype post-SCI. Gene expression of Agtr1a showed a trend for up-regulation at 2 months post-SCI; angiotensinogen was up-regulated at 2 month post-SCI in both genotypes. AngII receptor type 2 (Agtr2) was up- and down-regulated at 1 and 2 months post-SCI in WT mice, respectively, and Ang-(1-7) receptor (Mas) at 1 and 2 months post-SCI. Atrogin-1/MAFbx and MuRF1, atrophy markers, were not significantly up-regulated post-SCI. Our data show that lack of AT1a does not protect from cardiac atrophy post-SCI.

Depletion of angiotensin-converting enzyme 2 reduces brain serotonin and impairs the running-induced neurogenic response.

Physical exercise induces cell proliferation in the adult hippocampus in rodents. Serotonin (5-HT) and angiotensin (Ang) II are important mediators of the pro-mitotic effect of physical activity. Here, we examine precursor cells in the adult brain of mice lacking angiotensin-converting enzyme (ACE) 2, and explore the effect of an acute running stimulus on neurogenesis. ACE2 metabolizes Ang II to Ang-(1-7) and is essential for the intestinal uptake of tryptophan (Trp), the 5-HT precursor. In ACE2-deficient mice, we observed a decrease in brain 5-HT levels and no increase in the number of BrdU-positive cells following exercise. Targeting the Ang II/AT1 axis by blocking the receptor, or experimentally increasing Trp/5-HT levels in the brain of ACE2-deficient mice, did not rescue the running-induced effect. Furthermore, mice lacking the Ang-(1-7) receptor, Mas, presented a normal neurogenic response to exercise. Our results identify ACE2 as a novel factor required for exercise-dependent modulation of adult neurogenesis and essential for 5-HT metabolism.

Somatosensory BOLD fMRI reveals close link between salient blood pressure changes and the murine neuromatrix.

The neuromatrix, or "pain matrix", is a network of cortical brain areas which is activated by noxious as well as salient somatosensory stimulation. This has been studied in mice and humans using blood oxygenation level-dependent (BOLD) fMRI. Here we demonstrate that BOLD effects observed in the murine neuromatrix in response to salient somatosensory stimuli are prone to reflect mean arterial blood pressure (MABP) changes, rather than neural activity. We show that a standard electrostimulus typically used in murine somatosensory fMRI can induce substantial elevations in MABP. Equivalent drug-induced MABP changes - without somatosensory stimulation - evoked BOLD patterns in the neuromatrix strikingly similar to those evoked by electrostimulation. This constitutes a serious caveat for murine fMRI. The regional specificity of these BOLD patterns can be attributed to the co-localization of the neuromatrix with large draining veins. Based on these findings we propose a cardiovascular support mechanism whereby abrupt elevations in MABP provide additional energy supply to the neuromatrix and other essential brain areas in fight-or-flight situations.

High aminopeptidase A activity contributes to blood pressure control in ob/ob mice by AT2 receptor-dependent mechanism.

Obesity is assumed to be a major cause of human essential hypertension; however, the mechanisms responsible for weight-related increase in blood pressure (BP) are not fully understood. The prevalence of hypertension induced by obesity has grown over the years, and the role of the renin-angiotensin-aldosterone system (RAAS) in this process continues to be elucidated. In this scenario, the ob/ob mice are a genetic obesity model generally used for metabolic disorder studies. These mice are normotensive even though they present several metabolic conditions that predispose them to hypertension. Although the normotensive trait in these mice is associated with the poor activation of sympathetic nervous system by the lack of leptin, we demonstrated that ob/ob mice present massively increased aminopeptidase A (APA) activity in the circulation. APA enzyme metabolizes angiotensin (ANG) II into ANG III, a peptide associated with intrarenal angiotensin type 2 (AT2) receptor activation and induction of natriuresis. In these mice, we found increased ANG-III levels in the circulation, high AT2 receptor expression in the kidney, and enhanced natriuresis. AT2 receptor blocking and APA inhibition increased BP, suggesting the ANG III-AT2 receptor axis as a complementary BP control mechanism. Circulating APA activity was significantly reduced by weight loss independently of leptin, indicating the role of fat tissue in APA production. Therefore, in this study we provide new data supporting the role of APA in BP control in ob/ob mouse strain. These findings improve our comprehension about obesity-related hypertension and suggest new tools for its treatment.NEW & NOTEWORTHY In this study, we reported an increased angiotensin III generation in the circulation of ob/ob mice caused by a high aminopeptidase A activity. These findings are associated with an increased natriuresis found in these mice and support the role of renin-angiotensin-aldosterone system as additional mechanism regulating blood pressure in this genetic obese strain.

Dll4-Notch signaling determines the formation of native arterial collateral networks and arterial function in mouse ischemia models.

Arteriogenesis requires growth of pre-existing arteriolar collateral networks and determines clinical outcome in arterial occlusive diseases. Factors responsible for the development of arteriolar collateral networks are poorly understood. The Notch ligand Delta-like 4 (Dll4) promotes arterial differentiation and restricts vessel branching. We hypothesized that Dll4 may act as a genetic determinant of collateral arterial networks and functional recovery in stroke and hind limb ischemia models in mice. Genetic loss- and gain-of-function approaches in mice showed that Dll4-Notch signaling restricts pial collateral artery formation by modulating arterial branching morphogenesis during embryogenesis. Adult Dll4(+/-) mice showed increased pial collateral numbers, but stroke volume upon middle cerebral artery occlusion was not reduced compared with wild-type littermates. Likewise, Dll4(+/-) mice showed reduced blood flow conductance after femoral artery occlusion, and, despite markedly increased angiogenesis, tissue ischemia was more severe. In peripheral arteries, loss of Dll4 adversely affected excitation-contraction coupling in arterial smooth muscle in response to vasopressor agents and arterial vessel wall adaption in response to increases in blood flow, collectively contributing to reduced flow reserve. We conclude that Dll4-Notch signaling modulates native collateral formation by acting on vascular branching morphogenesis during embryogenesis. Dll4 furthermore affects tissue perfusion by acting on arterial function and structure. Loss of Dll4 stimulates collateral formation and angiogenesis, but in the context of ischemic diseases such beneficial effects are overruled by adverse functional changes, demonstrating that ischemic recovery is not solely determined by collateral number but rather by vessel functionality.

Neurolysin knockout mice generation and initial phenotype characterization.

The oligopeptidase neurolysin (EC 3.4.24.16; Nln) was first identified in rat brain synaptic membranes and shown to ubiquitously participate in the catabolism of bioactive peptides such as neurotensin and bradykinin. Recently, it was suggested that Nln reduction could improve insulin sensitivity. Here, we have shown that Nln KO mice have increased glucose tolerance, insulin sensitivity, and gluconeogenesis. KO mice have increased liver mRNA for several genes related to gluconeogenesis. Isotopic label semiquantitative peptidomic analysis suggests an increase in specific intracellular peptides in gastrocnemius and epididymal adipose tissue, which likely is involved with the increased glucose tolerance and insulin sensitivity in the KO mice. These results suggest the exciting new possibility that Nln is a key enzyme for energy metabolism and could be a novel therapeutic target to improve glucose uptake and insulin sensitivity.

Angiotensin deficient FVB/N mice are normotensive.

BACKGROUND AND PURPOSE: All previous rodent models lacking the peptide hormone angiotensin II (Ang II) were hypotensive. A mixed background strain with global deletion of the angiotensinogen gene was backcrossed to the FVB/N background (Agt-KO), a strain preferred for transgenic generation. Surprisingly, the resulting line turned out to be normotensive. Therefore, this study aimed to understand the unique blood pressure regulation of FVB/N mice without angiotensin peptides. EXPERIMENTAL APPROACH: Acute and chronic recordings of blood pressure (BP) in freely-moving adult mice were performed to establish baseline BP. The pressure responses to sympatholytic and sympathomimetic as well as a nitric oxide inhibitor and donor compounds were used to quantify the neurogenic tone and endothelial function. The role of the renal nerves on baseline BP maintenance was tested by renal denervation. Finally, further phenotyping was done by gene expression analysis, histology and measurement of metabolites in plasma, urine and tissues. KEY RESULTS: Baseline BP in adult FVB/N Agt-KO was unexpectedly unaltered. As compensatory mechanisms Agt-KO presented an increased sympathetic nerve activity and reduced endothelial nitric oxide production. However, FVB/N Agt-KO exhibited the renal morphological and physiological alterations previously found in mice lacking the production of Ang II including polyuria and hydronephrosis. The hypotensive effect of bilateral renal denervation was blunted in Agt-KO compared to wildtype FVB/N mice. CONCLUSION AND IMPLICATIONS: We describe a germline Agt-KO line that challenges all previous knowledge on BP regulation in mice with deletion of the classical RAS. This line may represent a model of drug-resistant hypertension because it lacks hypotension.

Genome Sequences of SARS-CoV-2 Strains from the Republic of Moldova.

The whole-genome sequences of 15 severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) strains from nasopharyngeal swab samples collected in the Republic of Moldova in June 2020 to September 2021 were determined. Little variability was observed in the early stages, when mostly clade 19A was circulating, followed by clade 20B. Later, multiple introductions of SARS-CoV-2 lineages B.1.1., B.1.1.7, and B.1.1.525 were detected. The B.1.1.7 lineage became predominant between December 2020 and June 2021, followed by the Delta variant.

Characterisation of the Antibody Response in Sinopharm (BBIBP-CorV) Recipients and COVID-19 Convalescent Sera from the Republic of Moldova.

The early availability of effective vaccines against SARS-CoV-2, the aetiologic cause of COVID-19, has been at the cornerstone of the global recovery from the pandemic. This study aimed to assess the antispike RBD IgG antibody titres and neutralisation potential of COVID-19 convalescent plasma and the sera of Moldovan adults vaccinated with the Sinopharm BBIBP-CorV vaccine. An IgG ELISA with recombinant SARS-CoV-2 spike RBD and two pseudovirus-based neutralisation assays have been developed to evaluate neutralising antibodies against SARS-CoV-2 in biosafety level 2 containment facilities. A significant moderate correlation was observed between IgG titres and the overall neutralising levels for each neutralisation assay (ρ = 0.64, p < 0.001; ρ = 0.52, p < 0.001). A separate analysis of convalescent and vaccinated individuals showed a higher correlation of neutralising and IgG titres in convalescent individuals (ρ = 0.68, p < 0.001, ρ = 0.45, p < 0.001) compared with vaccinated individuals (ρ = 0.58, p < 0.001; ρ = 0.53, p < 0.001). It can be concluded that individuals who recovered from infection developed higher levels of antispike RBD IgG antibodies. In comparison, the Sinopharm-vaccinated individuals produced higher levels of neutralising antibodies than convalescent plasma.

EVs vs. EVs: MSCs and Tregs as a source of invisible possibilities.

Extracellular vesicles (EVs) are produced by various cells and exist in most biological fluids. They play an important role in cell-cell signaling, immune response, and tumor metastasis, and also have theranostic potential. They deliver many functional biomolecules, including DNA, microRNAs (miRNA), messenger RNA (mRNA), long non-coding RNA (lncRNA), lipids, and proteins, thus affecting different physiological processes in target cells. Decreased immunogenicity compared to liposomes or viral vectors and the ability to cross through physiological barriers such as the blood-brain barrier make them an attractive and innovative option as diagnostic biomarkers and therapeutic carriers. Here, we highlighted two types of cells that can produce functional EVs, namely, mesenchymal stem/stromal cells (MSCs) and regulatory T cells (Tregs), discussing MSC/Treg-derived EV-based therapies for some specific diseases including acute respiratory distress syndrome (ARDS), autoimmune diseases, and cancer.

Quantifying the impact of gut microbiota on inflammation and hypertensive organ damage.

AIMS: Hypertension (HTN) can lead to heart and kidney damage. The gut microbiota has been linked to HTN, although it is difficult to estimate its significance due to the variety of other features known to influence HTN. In the present study, we used germ-free (GF) and colonized (COL) littermate mice to quantify the impact of microbial colonization on organ damage in HTN. METHODS AND RESULTS: 4-week-old male GF C57BL/6J littermates were randomized to remain GF or receive microbial colonization. HTN was induced by subcutaneous infusion with angiotensin (Ang) II (1.44 mg/kg/day) and 1% NaCl in the drinking water; sham-treated mice served as control. Renal damage was exacerbated in GF mice, whereas cardiac damage was more comparable between COL and GF, suggesting that the kidney is more sensitive to microbial influence. Multivariate analysis revealed a larger effect of HTN in GF mice. Serum metabolomics demonstrated that the colonization status influences circulating metabolites relevant to HTN. Importantly, GF mice were deficient in anti-inflammatory faecal short-chain fatty acids (SCFA). Flow cytometry showed that the microbiome has an impact on the induction of anti-hypertensive myeloid-derived suppressor cells and pro-inflammatory Th17 cells in HTN. In vitro inducibility of Th17 cells was significantly higher for cells isolated from GF than conventionally raised mice. CONCLUSION: The microbial colonization status of mice had potent effects on their phenotypic response to a hypertensive stimulus, and the kidney is a highly microbiota-susceptible target organ in HTN. The magnitude of the pathogenic response in GF mice underscores the role of the microbiome in mediating inflammation in HTN.

Growth retardation and altered autonomic control in mice lacking brain serotonin.

Serotonin synthesis in mammals is initiated by 2 distinct tryptophan hydroxylases (TPH), TPH1 and TPH2. By genetically ablating TPH2, we created mice (Tph2(-/-)) that lack serotonin in the central nervous system. Surprisingly, these mice can be born and survive until adulthood. However, depletion of serotonin signaling in the brain leads to growth retardation and 50% lethality in the first 4 weeks of postnatal life. Telemetric monitoring revealed more extended daytime sleep, suppressed respiration, altered body temperature control, and decreased blood pressure (BP) and heart rate (HR) during nighttime in Tph2(-/-) mice. Moreover, Tph2(-/-) females, despite being fertile and producing milk, exhibit impaired maternal care leading to poor survival of their pups. These data confirm that the majority of central serotonin is generated by TPH2. TPH2-derived serotonin is involved in the regulation of behavior and autonomic pathways but is not essential for adult life.

Sepsis induces interleukin 6, gp130/JAK2/STAT3, and muscle wasting.

BACKGROUND: Sepsis and inflammation can cause intensive care unit-acquired weakness (ICUAW). Increased interleukin-6 (IL-6) plasma levels are a risk factor for ICUAW. IL-6 signalling involves the glycoprotein 130 (gp130) receptor and the JAK/STAT-pathway, but its role in sepsis-induced muscle wasting is uncertain. In a clinical observational study, we found that the IL-6 target gene, SOCS3, was increased in skeletal muscle of ICUAW patients indicative for JAK/STAT-pathway activation. We tested the hypothesis that the IL-6/gp130-pathway mediates ICUAW muscle atrophy. METHODS: We sequenced RNA (RNAseq) from tibialis anterior (TA) muscle of cecal ligation and puncture-operated (CLP) and sham-operated wildtype (WT) mice. The effects of the IL-6/gp130/JAK2/STAT3-pathway were investigated by analysing the atrophy phenotype, gene expression, and protein contents of C2C12 myotubes. Mice lacking Il6st, encoding gp130, in myocytes (cKO) and WT controls, as well as mice treated with the JAK2 inhibitor AG490 or vehicle were exposed to CLP or sham surgery for 24 or 96 h. RESULTS: Analyses of differentially expressed genes in RNAseq (≥2-log2-fold change, P < 0.01) revealed an activation of IL-6-signalling and JAK/STAT-signalling pathways in muscle of septic mice, which occurred after 24 h and lasted at least for 96 h during sepsis. IL-6 treatment of C2C12 myotubes induced STAT3 phosphorylation (three-fold, P < 0.01) and Socs3 mRNA expression (3.1-fold, P < 0.01) and caused myotube atrophy. Knockdown of Il6st diminished IL-6-induced STAT3 phosphorylation (-30.0%; P < 0.01), Socs3 mRNA expression, and myotube atrophy. JAK2 (- 29.0%; P < 0.01) or STAT3 inhibition (-38.7%; P < 0.05) decreased IL-6-induced Socs3 mRNA expression. Treatment with either inhibitor attenuated myotube atrophy in response to IL-6. CLP-operated septic mice showed an increased STAT3 phosphorylation and Socs3 mRNA expression in TA muscle, which was reduced in septic Il6st-cKO mice by 67.8% (P < 0.05) and 85.6% (P < 0.001), respectively. CLP caused a loss of TA muscle weight, which was attenuated in Il6st-cKO mice (WT: -22.3%, P < 0.001, cKO: -13.5%, P < 0.001; WT vs. cKO P < 0.001). While loss of Il6st resulted in a reduction of MuRF1 protein contents, Atrogin-1 remained unchanged between septic WT and cKO mice. mRNA expression of Trim63/MuRF1 and Fbxo32/Atrogin-1 were unaltered between CLP-treated WT and cKO mice. AG490 treatment reduced STAT3 phosphorylation (-22.2%, P < 0.05) and attenuated TA muscle atrophy in septic mice (29.6% relative reduction of muscle weight loss, P < 0.05). The reduction in muscle atrophy was accompanied by a reduction in Fbxo32/Atrogin-1-mRNA (-81.3%, P < 0.05) and Trim63/MuRF1-mRNA expression (-77.6%, P < 0.05) and protein content. CONCLUSIONS: IL-6 via the gp130/JAK2/STAT3-pathway mediates sepsis-induced muscle atrophy possibly contributing to ICUAW.

Neutrophil-derived proteinase 3 induces kallikrein-independent release of a novel vasoactive kinin.

The kinin-forming pathway is activated on endothelial cells and neutrophils when high-molecular weight kininogen (HK) is cleaved by plasma kallikrein liberating bradykinin, a potent mediator of inflammation. Kinins are released during inflammatory conditions such as vasculitis, associated with neutrophil influx around blood vessels. Some patients with vasculitis have elevated plasma levels of neutrophil-derived proteinase 3 (PR3) and anti-PR3 Abs. This study investigated if neutrophil-derived PR3 could induce activation of the kinin pathway. PR3 incubated with HK, or a synthetic peptide derived from HK, induced breakdown and release of a novel tridecapeptide termed PR3-kinin, NH(2)-MKRPPGFSPFRSS-COOH, consisting of bradykinin with two additional amino acids on each terminus. The reaction was specific and inhibited by anti-PR3 and alpha(1)-antitrypsin. Recombinant wild-type PR3 incubated with HK induced HK breakdown, whereas mutated PR3, lacking enzymatic activity, did not. PR3-kinin bound to and activated human kinin B(1) receptors, but did not bind to B(2) receptors, expressed by transfected HEK293 cells in vitro. In human plasma PR3-kinin was further processed to the B(2) receptor agonist bradykinin. PR3-kinin exerted a hypotensive effect in vivo through both B(1) and B(2) receptors as demonstrated using wild-type and B(1) overexpressing rats as well as wild-type and B(2) receptor knockout mice. Neutrophil extracts from vasculitis patients and healthy controls contained comparable amounts of PR3 and induced HK proteolysis, an effect that was abolished when PR3 was immunoadsorbed. Neutrophil-derived PR3 can proteolyze HK and liberate PR3-kinin, thereby initiating kallikrein-independent activation of the kinin pathway.