Mast cell degranulation and bradykinin-induced angioedema - searching for the missing link.

Initiation of the bradykinin generation cascade is responsible for the occurrence of attacks in some types of angioedema without wheals. Hereditary angioedema due to C1 inhibitor deficiency (HAE-C1-INH) is one such clinical entity. In this paper, we explore the existing evidence that mast cells (MCs) degranulation may contribute to the activation of the kallikrein-kinin system cascade, followed by bradykinin formation and angioedema. We present the multidirectional effects of MC-derived heparin and other polyanions on the major components of the kinin-kallikrein system, particularly on the factor XII activation. Although, bradykinin- and histamine-mediated symptoms are distinct clinical phenomena, they share some common features, such as some similar triggers and a predilection to occur at sites where mast cells reside, namely the skin and mucous membranes. In addition, recent observations indicate a high incidence of hypersensitivity reactions associated with MC degranulation in the HAE-C1-INH patient population. However, not all of these can be explained by IgE-dependent mechanisms. Mast cell-related G protein-coupled receptor-X2 (MRGPRX2), which has recently attracted scientific interest, may be involved in the activation of MCs through a different pathway. Therefore, we reviewed MRGPRX2 ligands that HAE-C1-INH patients may be exposed to in their daily lives and that may affect MCs degranulation. We also discussed the known inter- and intra-individual variability in the course of HAE-C1-INH in relation to factors responsible for possible variability in the strength of the response to MRGPRX2 receptor stimulation. The above issues raise several questions for future research. It is not known to what extent a prophylactic or therapeutic intervention targeting the pathways of one mechanism (mast cell degranulation) may affect the other (bradykinin production), or whether the number of mast cells at a specific body site and their reactivity to triggers such as pressure, allergens or MRGPRX2 agonists may influence the occurrence of HAE-C1-INH attacks at that site.

Interaction of Angiotensin-(1-7) with kinins in the kidney circulation: Role of B1 receptors.

Changes in renal hemodynamics impact renal function during physiological and pathological conditions. In this context, renal vascular resistance (RVR) is regulated by components of the Renin-Angiotensin System (RAS) and the Kallikrein-Kinin System (KKS). However, the interaction between these vasoactive peptides on RVR is still poorly understood. Here, we studied the crosstalk between angiotensin-(1-7) and kinins on RVR. The right kidneys of Wistar rats were isolated and perfused in a closed-circuit system. The perfusion pressure and renal perfusate flow were continuously monitored. Ang-(1-7) (1.0-25.0 nM) caused a sustained, dose-dependent reduction of relative RVR (rRVR). This phenomenon was sensitive to 10 nM A-779, a specific Mas receptor (MasR) antagonist. Bradykinin (BK) promoted a sustained and transient reduction in rRVR at 1.25 nM and 125 nM, respectively. The transient effect was abolished by 4 µM des-Arg9-Leu8-bradykinin (DALBK), a specific kinin B1 receptor (B1R) antagonist. Accordingly, des-Arg9-bradykinin (DABK) 1 µM (a B1R agonist) increased rRVR. Interestingly, pre-perfusion of Ang-(1-7) changed the sustained reduction of rRVR triggered by 1.25 nM BK into a transient effect. On the other hand, pre-perfusion of Ang-(1-7) primed and potentiated the DABK response, this mechanism being sensitive to A-779 and DALBK. Binding studies performed with CHO cells stably transfected with MasR, B1R, and kinin B2 receptor (B2R) showed no direct interaction between Ang-(1-7) with B1R or B2R. In conclusion, our findings suggest that Ang-(1-7) differentially modulates kinin's effect on RVR in isolated rat kidneys. These results help to expand the current knowledge regarding the crosstalk between the RAS and KKS complex network in RVR.

Recent advances in the discovery and development of drugs targeting the kallikrein-kinin system.

BACKGROUND: The kallikrein-kinin system is a key regulatory cascade involved in blood pressure maintenance, hemostasis, inflammation and renal function. Currently, approved drugs remain limited to the rare disease hereditary angioedema. However, growing interest in this system is indicated by an increasing number of promising drug candidates for further indications. METHODS: To provide an overview of current drug development, a two-stage literature search was conducted between March and December 2023 to identify drug candidates with targets in the kallikrein-kinin system. First, drug candidates were identified using PubMed and Clinicaltrials.gov. Second, the latest publications/results for these compounds were searched in PubMed, Clinicaltrials.gov and Google Scholar. The findings were categorized by target, stage of development, and intended indication. RESULTS: The search identified 68 drugs, of which 10 are approved, 25 are in clinical development, and 33 in preclinical development. The three most studied indications included diabetic retinopathy, thromboprophylaxis and hereditary angioedema. The latter is still an indication for most of the drug candidates close to regulatory approval (3 out of 4). For the emerging indications, promising new drug candidates in clinical development are ixodes ricinus-contact phase inhibitor for thromboprophylaxis and RZ402 and THR-149 for the treatment of diabetic macular edema (all phase 2). CONCLUSION: The therapeutic impact of targeting the kallikrein-kinin system is no longer limited to the treatment of hereditary angioedema. Ongoing research on other diseases demonstrates the potential of therapeutic interventions targeting the kallikrein-kinin system and will provide further treatment options for patients in the future.

Potential Pathways and Pathophysiological Implications of Viral Infection-Driven Activation of Kallikrein-Kinin System (KKS).

Microcirculatory and coagulation disturbances commonly occur as pathological manifestations of systemic viral infections. Research exploring the role of the kallikrein-kinin system (KKS) in flavivirus infections has recently linked microvascular dysfunctions to bradykinin (BK)-induced signaling of B2R, a G protein-coupled receptor (GPCR) constitutively expressed by endothelial cells. The relevance of KKS activation as an innate response to viral infections has gained increasing attention, particularly after the reports regarding thrombogenic events during COVID-19. BK receptor (B2R and B1R) signal transduction results in vascular permeability, edema formation, angiogenesis, and pain. Recent findings unveiling the role of KKS in viral pathogenesis include evidence of increased activation of KKS with elevated levels of BK and its metabolites in both intravascular and tissue milieu, as well as reports demonstrating that virus replication stimulates BKR expression. In this review, we will discuss the mechanisms triggered by virus replication and by virus-induced inflammatory responses that may stimulate KKS. We also explore how KKS activation and BK signaling may impact virus pathogenesis and further discuss the potential therapeutic application of BKR antagonists in the treatment of hemorrhagic and respiratory diseases.

Kinin-kallikrein system: New perspectives in heart failure.

Heart failure (HF) is a pervasive clinical challenge characterized by compromised cardiac function and reduced quality of life. The kinin-kallikrein system (KSS), a multifaceted peptide cascade, has garnered substantial attention due to its potential role in HF. Through activation of B1 and/or B2 receptors and downstream signaling, kinins modulate various physiological processes, including inflammation, coagulation, pain, blood pressure control, and vascular permeability. Notably, aberrations in KKS components have been linked to HF risk. The elevation of vasodilatory bradykinin (BK) due to kallikrein activity reduces preload and afterload, while concurrently fostering sodium reabsorption inhibition. However, kallikrein's conversion of prorenin to renin leads to angiotensinsII upregulation, resulting in vasoconstriction and fluid retention, alongside increased immune cell activity that fuels inflammation and cardiac remodeling. Importantly, prolonged KKS activation resulting from volume overload and tissue stretch contributes to cardiac collagen loss. The conventional renin-angiotensin-aldosterone system (RAAS) inhibitors used in HF management may inadvertently intensify KKS activity, exacerbating collagen depletion and cardiac remodeling. It is crucial to balance the KKS's role in acute cardiac damage, which may temporarily enhance function and metabolic parameters against its detrimental long-term effects. Thus, KKS blockade emerges as a promising strategy to impede HF progression. By attenuating the link between immune system function and tissue damage, KKS inhibition can potentially reduce cardiac remodeling and alleviate HF symptoms. However, the nuanced roles of BK in various acute conditions necessitate further investigation into the sustained benefits of kallikrein inhibitors in patients with chronic HF.

¿Es el sistema calicreína/quinina renal un factor modulador de la calciuria? / Is the renal kallikrein-kinin system a factor that modulates calciuria?

La reabsorción tubular de calcio es uno de los principales factores que determinan la concentración sérica de calcio y su excreción urinaria. El túbulo contorneado distal y conector es donde se produce la regulación fina de la calciuria. A ese nivel se encuentra el canal epitelial de Ca (TRPV5), que es el paso limitante en el transporte transcelular de Ca. La presencia dinámica del canal TRPV5 en la superficie de la célula tubular está mediada por un proceso de reciclado endosómico. Distintos factores intrarrenales intervienen en la fijación del canal de calcio en la membrana aplical, entre ellos la hormona antienvejecimiento klotho y la calicreína tisular (CT). Ambas proteínas son sintetizadas en el túbulo distal y secretadas en el fluido tubular. La calicreína tisular estimula la reabsorción activa de calcio por vía del receptor de bradiquinina tipo 2 que compromete la activación del of TRPV5 por vía de la protein cinasa C. Los ratones deficientes en CT muestran hipercalciuria de origen renal comparable a la pérdida de calcio que se observa en los ratones knockout para el TRPV5. Existe un polimorfismo con pérdida de función del gen de la CT humana denominado R53H (alelo H) que produce una gran disminución de la actividad enzimática. La presencia del alelo H, por lo menos en la población japonesa, parece ser frecuente (24%). Estos individuos tiene una tendencia a excreción más alta de calcio y sodio en orina que se manifiesta más durante la infusión de furosemida. En el futuro habrá que estudiar si la manipulación del sistema calicreína-quinina renal permite corregir la hipercalciuria idiopática con fármacos diferentes a los diuréticos tiazídicos (AU)

Renal tubular calcium reabsorption is one of the principal factors that determine serum calcium concentration and calcium excretion. Calcium excretion is regulated by the distal convoluted tubule and connecting tubule, where the epithelial calcium channel TRPV5 can be found, which limits the rate of transcellular calcium transport. The dynamic presence of the TRPV5 channel on the surface of the tubular cell is mediated by an endosomal recycling process. Different intrarenal factors are involved in calcium channel fixation in the apical membrane, including the anti-ageing hormone klotho and tissue kallikrein (TK). Both proteins are synthesised in the distal tubule and secreted in the tubular fluid. TK stimulates active calcium reabsorption through the bradykinin receptor B2 that compromises TRPV5 activation through the protein kinase C pathway. TK-deficient mice show hypercalciuria of renal origin comparable to that seen in TRPV5 knockout mice. There is a polymorphism with loss of function of the human TK gene R53H (allele H) that causes a marked decrease in enzymatic activity. The presence of the allele H seems to be common at least in the Japanese population (24%). These individuals have a tendency to greater calcium and sodium excretion in urine that is more evident during furosemide infusion. Future studies should analyse if manipulating the renal kallikrein-kinin system can correct idiopathic hypercalciuria with drugs other than thiazide diuretics (AU)

Human Tissue Kallikrein Activity in Angiographically Documented Chronic Stable Coronary Artery Disease / Atividade da Calicreína Tecidual Humana na Doença Coronariana Crônica Estável Angiograficamente Documentada

AbstractBackground:Human tissue kallikrein (hK1) is a key enzyme in the kallikrein–kinin system (KKS). hK1-specific amidase activity is reduced in urine samples from hypertensive and heart failure (HF) patients. The pathophysiologic role of hK1 in coronary artery disease (CAD) remains unclear.Objective:To evaluate hK1-specific amidase activity in the urine of CAD patientsMethods:Sixty-five individuals (18–75 years) who underwent cardiac catheterism (CATH) were included. Random midstream urine samples were collected immediately before CATH. Patients were classified in two groups according to the presence of coronary lesions: CAD (43 patients) and non-CAD (22 patients). hK1 amidase activity was estimated using the chromogenic substrate D-Val-Leu-Arg-Nan. Creatinine was determined using Jaffé’s method. Urinary hK1-specific amidase activity was expressed as µM/(min · mg creatinine) to correct for differences in urine flow rates.Results:Urinary hK1-specific amidase activity levels were similar between CAD [0.146 µM/(min ·mg creatinine)] and non-CAD [0.189 µM/(min . mg creatinine)] patients (p = 0.803) and remained similar to values previously reported for hypertensive patients [0.210 µM/(min . mg creatinine)] and HF patients [0.104 µM/(min . mg creatinine)]. CAD severity and hypertension were not observed to significantly affect urinary hK1-specific amidase activity.Conclusion:CAD patients had low levels of urinary hK1-specific amidase activity, suggesting that renal KKS activity may be reduced in patients with this disease.

ResumoFundamento:A calicreína tecidual humana (hK1) é enzima-chave do sistema calicreína-cinina (SCC). A atividade amidásica da hK1 está reduzida na urina de pacientes com hipertensão e insuficiência cardíaca (IC); seu papel na doença arterial (DAC) coronariana ainda não está esclarecido.Objetivo:Avaliar a atividade amidásica da hK1 na urina de pacientes com DAC.Métodos:Sessenta e cinco indivíduos (18 a 75 anos) que se submeteram ao cateterismo cardíaco (CAT) coletaram amostra do jato médio de urina imediatamente antes do CAT. Baseando-se na presença de lesões coronarianas, os pacientes eram classificados em dois grupos: DAC (43 pacientes) e sem DAC (22 indivíduos). A atividade amidásica da hK1 foi estimada com o substrato cromogênico D-Val-Leu-Arg-Nan. Creatinina foi determinada pelo método de Jaffé. A atividade amidásica específica da hK1 urinária foi expressa em µM/(min . mg de creatinina) para corrigir diferenças no fluxo urinário.Resultados:A atividade amidásica da hK1 urinária foi semelhante entre os pacientes com DAC [0,146 µM/(min . mg de creatinina)] e aqueles sem DAC [0,189 µM/(min . mg de creatinina)] (p = 0,803), e permaneceu entre os baixos valores previamente publicados para pacientes com hipertensão primária [0,210 µM/(min . mg de creatinina)] e para aqueles com IC [0,104 µM/(min . mg de creatinina)], respectivamente. Nenhum efeito estatisticamente significativo da gravidade da DAC e da hipertensão sobre a atividade amidásica da hK1 urinária foi observado.Conclusão:A atividade amidásica da hK1 na urina estava reduzida nos pacientes com DAC, o que pode sugerir que a atividade do SCC renal esteja reduzida nessa doença.

Rol del sistema kallicreína kinina y su interrelación con sistemas vasoactivos durante la preñez / Role of the kallikrein kinin system and its interrelationship with vasoactive systems in pregnancy

La hiperfiltración glomerular y el aumento de la reabsorción de sodio son factores fundamentales para el desarrollo de la unidad feto placentaria. Dichos factores resultan de adaptaciones hemodinámicas y renales en las que participan sistemas vasoactivos. Se pudo demostrar en ratas que la activación del sistema kallicreína kinina (SKK) precede a la instalación de la hiperfiltración glomerular, dado que su inhibición por aprotinina previene el aumento del filtrado glomerular. Además, la inhibición individual o asociada de los efectores específicos del SKK, las prostaglandinas (PGs) y el óxido nítrico (ON), confirman la dependencia del filtrado glomerular del SKK durante la preñez. Encontramos también que el sistema renina angiotensina (SRA) participa en la generación de la hiperfiltración glomerular dado que ésta es afectada por la administración de bloqueantes del SRA. La inhibición máxima sobre el pico de hiperfiltración se obtuvo con el bloqueo de ambos sistemas (SKK y SRA). Además, estrategias para alterar la hiperfiltración glomerular y la reabsorción de sodio de la preñez evidenciaron alteraciones en el desarrollo de la unidad feto placentaria, menor número de crías, mayor cantidad de reabsorciones intrauterinas y retardo en el crecimiento. El tratamiento combinado de inhibidores del SKK asociados a bloqueantes del SRA o de óxido nítrico mostraron los mayores efectos. En consecuencia, demostramos que el SKK juega un rol central en los fenómenos de adaptación que acompañan la preñez normal. La interrelación del SKK con varios sistemas vasoactivos parecería formar una red que participa en las adaptaciones hemodinámicas para un adecuado desarrollo de la gestación y de la unidad feto-placentaria.

Glomerular hyperfiltration and increased sodium reabsorption are key factors for the development of the fetus and placenta in pregnancy. These adjustments result from hemodynamic and renal factors involving vasoactive systems. It was demonstrated in rats that activation of KKS precedes the installation of glomerular hyperfiltration as aprotinin prevents the increase in glomerular filtration. In addition, individual or associated inhibition of specific kallikrein kinin system effectors, prostaglandins (PGs) and nitric oxide (NO), confirm the glomerular filtration rate dependence of KKS during pregnancy. It was also found that the renin-angiotensin system (RAS) contributes to glomerular hyperfiltration as this is affected by the administration of RAS blockers. The peak of hyperfiltration maximum inhibition was obtained by the blockade of both systems (KKS and RAS). In addition, strategies used to alter the glomerular hyperfiltration and increased sodium reabsorption during pregnancy, showed abnormalities in the development of the fetus and placenta, fewer offspring, more fetus resorptions and intrauterine growth retardation. KKS inhibitors associated with RAS or nitric oxide blockers showed the greatest impact. As a consequence, it was demonstrated that KKS plays a central role in the adaptation phenomenom that accompanies normal pregnancy. The interplay of KKS with several vasoactive systems, seem to arrange a network involved in the hemodynamic adaptations to allow the proper development of pregnancy and the fetus and placenta.

Portal hypertensive response to kinin

Portal hypertension is the most common complication of chronic liver diseases, such as cirrhosis. The increased intrahepatic vascular resistance seen in hepatic disease is due to changes in cellular architecture and active contraction of stellate cells. In this article, we review the historical aspects of the kallikrein-kinin system, the role of bradykinin in the development of disease, and our main findings regarding the role of this nonapeptide in normal and experimentalmodels of hepatic injury using the isolated rat liver perfusion model (mono and bivascular) and isolated liver cells. We demonstrated that: 1) the increase in intrahepatic vascular resistance induced by bradykinin is mediated by B2 receptors, involving sinusoidal endothelial and stellate cells, and is preserved in the presence of inflammation, fibrosis, and cirrhosis; 2) the hepatic arterial hypertensive response to bradykinin is calcium-independent and mediated by eicosanoids; 3) bradykinin does not have vasodilating effect on the pre-constricted perfused rat liver; and, 4) after exertion of its hypertensive effect, bradykinin is degraded by angiotensin converting enzyme. In conclusion, the hypertensive response to BK is mediated by the B2 receptor in normal and pathological situations. The B1 receptor is expressed more strongly in regenerating and cirrhotic livers, and its role is currently under investigation.

Hipertensão portal é a complicação mais comum das doenças crônicas do fígado, tais como cirrose. A resistência intravascular aumentada observada na doença hepática é devida a alterações na arquitetura celular e contração ativa das células estreladas. Neste trabalho revisamos aspectos históricos do estudo do sistema calicreína-cinina e os resultados de nossos estudos do papel deste nonapeptídeo no controle do tono vascular intra-hepático em condições normais e modelos experimentais de agressão hepática usando a perfusão de fígado isolado de rato (mono e bivascular) e células hepáticas isoladas. Nós demonstramos que: 1) o aumento da resistência vascular intrahepática induzido pela bradicinina é mediado por receptores B2, envolve a participação de células endoteliais sinusoidais e células estreladas e não é alterada pela presença de inflamação, fibrose ou cirrose; 2) a resposta hipertensiva induzida pela bradicinina no sistema arterial hepático é cálcio-independente emediada por eicosanóides; 3) bradicinina não tem efeito dilatador na circulação intra-hepática; 4) após exercer efeito vasoconstritor intra-hepático, a bradicinina é degradada pela enzima conversora de angiotensina. Em conclusão, a resposta hipertensiva à bradicinina é mediada pelo receptor B2 em condições normais e patológicas. Receptor B1 é expresso mais fortemente nos fígados em regeneração e cirróticos e seu papel está sob investigação.

El kininógeno de alto peso molecular: su participación en la respuesta inflamatoria y en la angiogénesis. Propiedades y posible aplicación terapéutica / High molecular weight kininogen in inflammation and angiogenesis: a review of its properties and therapeutic applications

The plasma kallikrein-kinin system (KKS) participates in the pathogenesis of inflammatory reactions involved in cellular injury, coagulation, fibrinolysis, kinin formation, complement activation, cytokine secretion and release of proteases. It has been shown that KKS activation in the systemic inflammatory response syndrome results in decrease of its component plasma proteins. Similar changes have been documented in diabetes, sepsis, children with vasculitis, allograft rejection, disseminated intravascular coagulation, patients with recurrent pregnancy losses, hereditary angioedema, adult respiratory distress syndrome and coronary artery disease. Direct involvement of the KKS in the pathogenesis of experimental acute arthritis and acute and chronic enterocolitis has been documented by previous studies from our laboratory using experimental animal models. It has been found that in HK deficient Lewis rats, experimental IBD was much less severe. We showed a genetic difference in kininogen structure between resistant Buffalo and susceptible Lewis rats, which results in accelerated cleavage of HK and it is responsible for the susceptibility to the inflammatory process in the Lewis rats. It has been demostrated that therapy with a specific plasma kallikrein inhibitor (P8720) modulated the experimental enterocolitis, arthritis and systemic inflammation. Furthermore, it has been shown that a bradykinin 2 receptor (B2R) antagonist attenuates the inflammatory changes in the same animal model. We have showed that a monoclonal antibody targeting HK decreases angiogénesis and arrests tumor growth in a syngeneic animal model. In summary, these results indicate that the plasma KKS plays a central role in the pathogenesis of chronic intestinal inflammation, arthritis and angiogenesis.

Se ha demostrado la participación del sistema plasmático de kalikreína-kininas (KKS) en el proceso inflamatorio, el cual incluye reacciones de daño celular, coagulación y fibrinólisis, formación de kininas, activación del complemento, secreción de citoquinas y liberación de proteasas. El KKS se encuentra activado en el síndrome de respuesta inflamatoria sistémica con una disminución en la concentración plasmática de las proteínas que lo constituyen. También se ha demostrado una activación similar en la diabetes, choque séptico, vasculitis en infantes, enfermedad injerto-huésped, coagulación intravascular diseminada, pacientes con abortos de repetición, angioedema hereditario, el síndrome de estrés respiratorio del adulto y enfermedad coronaria arterial. Mediante el uso de modelos animales experimentales, nuestro laboratorio ha demostrado una participación directa del KKS en la patogénesis de la artritis experimental aguda y la enterocolitis aguda y crónica. Se ha demostrado que en la rata tipo Lewis, cuando es deficiente de kininógeno de alto peso molecular (HK), la enfermedad inflamatoria intestinal es menos severa comparada con la presentada en ratas con niveles normales de HK como la Buffalo. Nosotros mostramos una diferencia entre el gene que codifica la molécula del kininógeno de la rata tipo Buffalo (resistentes) y Lewis (susceptibles), que resulta en un incremento de la actividad proteolítica de kalikreína sobre su substrato HK, lo cual predispone a las ratas Lewis al desarrollo de la enfermedad inflamatoria crónica. Se ha demostrado una disminución en las manifestaciones inflamatorias sistémicas de la enterocolitis y artritis experimental mediante el uso de un inhibidor específico de la kalikreína (P8720). Además, el antagonista del receptor 2 de la bradikinina (BR2) atenuó los cambios inflamatorios en el mismo modelo animal. Asimismo, se ha demostrado que las ratas Lewis deficientes de kininógeno desarrollaron inflamación intestinal sistémica menos severa. Mediante el uso del anticuerpo monoclonal C11C1 contra HK se logró una disminución de la angiogenesis y, consecuentemente, el crecimiento tumoral. En conclusión, los resultados demuestran que el sistema plasmático de KKS desempeña un papel preponderante en la patogénesis de la artritis reumatoide, la enfermedad intestinal crónica y en el proceso angiogénico.

The balance between the production of tumor necrosis factor-alpha and interleukin-10 determines tissue injury and lethality during intestinal ischemia and reperfusion

A major goal in the treatment of acute ischemia of a vascular territory is to restore blood flow to normal values, i.e. to "reperfuse" the ischemic vascular bed. However, reperfusion of ischemic tissues is associated with local and systemic leukocyte activation and trafficking, endothelial barrier dysfunction in postcapillary venules, enhanced production of inflammatory mediators and great lethality. This phenomenon has been referred to as "reperfusion injury" and several studies demonstrated that injury is dependent on neutrophil recruitment. Furthermore, ischemia and reperfusion injury is associated with the coordinated activation of a series of cytokines and adhesion molecules. Among the mediators of the inflammatory cascade released, TNF-alpha appears to play an essential role for the reperfusion-associated injury. On the other hand, the release of IL-10 modulates pro-inflammatory cytokine production and reperfusion-associated tissue injury. IL-1beta, PAF and bradykinin are mediators involved in ischemia and reperfusion injury by regulating the balance between TNF-alpha and IL-10 production. Strategies that enhance IL-10 and/or prevent TNF-alpha concentration may be useful as therapeutic adjuvants in the treatment of the tissue injury that follows ischemia and reperfusion.

Estudo da regulação da expressão do gene da carboxipeptidase M de camundongo / Expression regulation studies of the carboxypeptidase M gene in mouse

A caracterização da estrutura molecular da carboxipeptidase M humana originou os estudos da enzima em camundongos. Utilizando análise computacional a estrutura do gene em camundongos foi determinada comparando a seqüência de humanos no banco de dados genômico de camundongos. As duas seqüências apresentaram 82 por cento de similaridade entre elas. Foram determinados todos os fatores envolvidos na caracterização do RNAm como tamanho de exons e introns, região de catálise, região hidrofóbica, peptídeo sinal, sítios de glicosilação e glutâmicos catalíticos. A análise computacional proporcionou também a observação de um EST o qual também se tomou objeto de estudos. Após serem definidos todos os fatores pertencentes ao gene iniciamos os experimentos com a demonstração da atividade luciferase para os promotores da CPM e do EST. A análise foi feita em três diferentes tipos celulares para evidenciar a existência de atividade promotora e sua diferente regulação nessas linhagens. O EST foi clonado e transfectado em células para dosagem fluorimetrica de sua atividade carboxipeptidase básica. A técnica de PCR em tempo real foi utilizada para obtenção dos resultados de expressão gênica relativa tanto da carboxipeptidase M como do EST em camundongos nas fases embrionária, neonatal e adulta. Com base nos dados obtidos pudemos concluir a estrutura da carboxipeptidase M e do EST encontrado, sua regulação transcricional e padrão de expressão nos diversos RNAs extraídos de tecidos de camundongos.

Papel do sistema calicreína-cinina no desenvolvimento de câncer de mama em ratos TGRhKLK1 / The role of kallikrein-kinin System in the development of breast cancer in rats TGRhKLK1

As calicreínas humanas compreendem a maior família de serino endopeptidas, homologas num único cluster no genoma humano. O primeiro membro desta família f identificado na década de 30s como uma proteína abundante no pâncreas sendo chama( calicreína tecidual. Posteriormente foram descobertos o antígeno prostático especifico (PSA) ou hK3 e a calicreína plasmática hK2. Recentemente foram descritos outros l2 genes de serino proteases relacionados com a família de genes da calicreína os quais se co-localizam no cromossomo 19q13.4 e apresentam similaridade estrutural e de seqüências aos três primeiros genes descritos. Recentemente a família hKLK foi envolvida nos processos de tumorigênes crescimento celular, angiogênese, invasão e metástase através do processamento proteolítico de proteínas que se ligam a fatores de crescimento, ativação de fatores c crescimento e outras proteases, liberação de fatores angiogênicos ou anti-angiogênicos degradação de componentes da matriz extracelular. Além disso muitas das novas "calicreínas" estão sendo utilizadas como promissores marcadores de diagnostico prognostico em câncer. No presente estudo nós utilizamos o modelo de ratos transgênicos com superexpressão do gene humano da calicreína tecidual 1 (TGRhKLK1) para avaliar os efeitos na iniciação, proliferação e desenvolvimento de câncer de mama induzido por um carcinógeno químico, o DMBA. Na literatura não existem estudos in vivo provando participação direta de hKLK1 no processo neoplásico, apesar da grande quantidade de informação existente envolvendo a participação dos restantes membros da família das calicreínas no processo tumoral. Nesse trabalho foi demonstrado que a superexpressão de hKLK1 induz a ativação do início da transformação neoplásica e promove o crescimento tumores de mama induzidos por DMBA...

Estudo da interação de proteínas dos sistema calicreína-cininas com glicosaminoglicanos / Study of the interaction of Kallikrein-Kinin system proteins with glycosaminoglycans

A calicreína plasmática humana (huPK) é uma serinoendopeptidas envolvida em muitos processos biológicos, entre eles o sistema calicreína-cinina. Em relação a esse sistema, tem sido mostrado que a procalicreína (PK), quando ligada à membrana de células endoteliais por meio do HK, é ativada à calicreína, resultando na liberação de bradicinina (BK), um potente mediador da resposta inflamatória. A liberação de BK pode ser influenciada por muitos compostos e objetivo desse trabalho foi investigar o efeito de glicosaminoglicanos na hidrólise do HK pela huPK in vitro, na conformação do HK e da huPK, na ligação do HK da PK à superfície e matriz extracelular de células endoteliais e em um modelo c inflamação in vivo. Para isso, huPK foi incubada com HK, na ausência e presença de diferentes glicosaminoglicanos. A liberação de BK, em diferentes tempos, foi medida por radioimunoensaio e as constantes catalíticas foram calculadas. Dermatam sulfato de atum e bovino reduziram a eficiência catalítica da huPK sobre o HK (controle = 4.1 x 104 M-¹ s-¹) em 80 por cento e 68 por cento, respectivamente. Então, o efeito do dermatam sulfato bovino (DSb) na resposta inflamatória foi estudado num modelo de edema de pata de rato induzido por carragenina. O volume da pata do rato foi medido com o auxílio de um pletismômetro, de 1 a 4 horas após administração da carragenina. O DSb reduziu significativamente (p< 0.05) a resposta inflamatória na primeira e segunda hora de medida, em 24 por cento e 28 por cento, respectivamente. A interação da huPK com um glicosaminoglicano foi verificada por cromatografia de afinidade em Heparina-Sepharose. A huPK apresentou uma alta afinidade pela heparina, já que foi eluída da coluna com NaCl 0,5 M. A influência dos glicosaminoglicanos na estrutura secundária da huPK e do HK foi analisada por dicroísmo circular. Apenas os condroitim 4- e 6-sulfato modificaram essas estruturas...

Papel de mediadores endógenos na fisiopatologia da cirrose hepática / The role of endogenous mediators in the pathophysiology of hepatic cirrhosis

A cirrose hepática é o evento final das doenças hepáticas crônicas em pacientes adultos e pediátricos. Apesar de muitos estudos em relação ao desenvolvimento da fibrose hepática, o entendimento de sua etiopatogênese e fisiopatologia continua obscuro. Diversos mediadores, tais como componentes do sistema renina-angiotensina, o sistema nervoso simpático, a vasopressina, os peptideos natriuréticos, o sistema calicreína-cinina, a endotelina e o óxido nítrico podem estar envolvidos nesses mecanismos. Além disso, a abordagem terapêutica dos pacientes hepatopatas ainda é bastante limitada, contribuindo para o prognóstico reservado. Este artigo de revisão pretende discutir algumas evidências clínicas e experimentais relativas à participação destes mediadores endógenos na fisiopatologia da cirrose hepática.

The hepatic cirrhosis is the final event of many chronic hepatic diseases in adult and pediatric patients. In spite of many studies regarding the development of fibrosis, the understanding of its etio- and physiopathology remains obscure. Several mediators may be involved in these mechanisms: renin-angiotensin system, sympathetic nervous system, vasopressin, natriuretic peptides, kallicreinkynin system, endothelin, and nitric oxide. Furthermore, the therapeutic approach of the patients with chronic hepatic diseases is still limited, contributing to a bad outcome. Some clinical and experimental evidences regarding the participation of these endogeneous mediators in the physiopathology of hepatic cirrhosis are discussed.

Substratos e inibidores para a calicreína tissular humana / Human tissue kallikrein substrates and inhibitors

Este trabalho focaliza o sistema calicreína cinina, do ponto de vista da - interaçao da calicreína tissular humana com seu inibidor proteico do tipo serpina, denominado calistatina. Para tanto, o mapeamento dos determinantes de especificidade da calicreína tissular humana foram explorados através do uso de substratos peptídicos sintéticos com supressao intramolecular de fluorescência. Este estudo, associado a informaçoes anteriores a cerca da especificidade desta enzima sobre seqüências derivadas do cininogênio humano foram capazes de gerar inibidores competitivos com alta afinidade, contendo inclusive aminoácidos nao naturais. Além disso, o estudo da calistatina também foi abordado, procurando-se determinar alguns dos fatores que podem regular sua atividade fisiológica, como a interaçao com heparina e a sua destruiçao por outras proteases. Os resultados apresentados indicam que o centro reativo da calistatina foi capaz de servir de molde para a síntese de inibidores eficazes para a calicreína tissular humana e que pequenos peptídeos, contendo em sua seqüência aminoácido nao naturais apresentam um caminho potencial para o desenvolvimento de inibidores peptídicos específicos. Estudamos ainda a interaçao da heparina com calistatina e apresentamos evidencias do mecanismo de sua perda de funçao inibitória do complexo. Finalmente, abordamos a da calistatina e da antiquimotripsina pela catepsina D, que além evidenciar um possível papel desta enzima no controle destas serpinas, coletamos informaçoes relevantes sobre a especificidade da catepsina D

Regulation of the kinin receptors after induction of myocardial infarction: a mini-review

It is well known that the responses to vasoactive kinin peptides are mediated through the activation of two receptors termed bradykinin receptor B1 (B1R) and B2 (B2R). The physiologically prominent B2R subtype has certainly been the subject of more intensive efforts in structure-function studies and physiological investigations. However, the B1R activated by a class of kinin metabolites has emerged as an important subject of investigation within the study of the kallikrein-kinin system (KKS). Its inducible character under stress and tissue injury is therefore a field of major interest. Although the KKS has been associated with cardiovascular regulation since its discovery at the beginning of the last century, less is known about the B1R and B2R regulation in cardiovascular diseases like hypertension, myocardial infarction (MI) and their complications. This mini-review will summarize our findings on B1R and B2R regulation after induction of MI using a rat model. We will develop the hypothesis that differences in the expression of these receptors may be associated with a dual pathway of the KKS in the complex mechanisms of myocardial remodeling.