Minireview: functional roles of tissue kallikrein, kinins, and kallikrein-related peptidases in lung cancer.

Despite campaigns and improvements in detection and treatment, lung cancer continues to increase worldwide and represents a major public health problem. One approach to treating patients suffering from lung cancer is to target surface receptors overexpressed on tumor cells, such as GPCR-family kinin receptors, and proteases that control tumor progression, such as kallikrein-related peptidases (KLKs). These proteases have been visualized in recent years due to their contribution to the progression of cancers, such as prostate and ovarian cancer, facilitating the invasive and metastatic capacity of tumor cells in these tissues. In fact, KLK3 is the specific prostate antigen, the only tissue-specific biomarker used to diagnose this malignancy. In lung cancer to date, evidence indicates that KLK5, KLK6, KLK8, KLK11, and KLK14 are the major peptidases regulated and involved in its progression. The expression levels of KLKs in this neoplasm are modulated by the secretome of the different cell types present in the tumor microenvironment, the cancer subtype and the tumor stage, among others. Considering the multiple functions of kinin receptors and KLKs, this review highlights their roles, even considering the SARS-CoV-2 effects. Since lung cancer is often diagnosed in advanced stages, our efforts should focus on early diagnosis, validating for example specific KLKs, especially in high-risk populations such as smokers and people exposed to carcinogenic fumes, oil fields, and contaminated workplaces, unexplored fields to investigate. Furthermore, their modulation could be considered as a promising approach in lung cancer therapeutics.

The family of kallikrein-related peptidases and kinin peptides as modulators of epidermal homeostasis.

The epidermis is the outermost skin layer and is part of one of the largest organs in the body; it is supported by the dermis, a network of fibrils, blood vessels, pilosebaceous units, sweat glands, nerves, and cells. The skin as a whole is a protective shield against numerous noxious agents, including microorganisms and chemical and physical factors. These functions rely on the activity of multiple growth factors, peptide hormones, proteases, and specific signaling pathways that are triggered by the activation of distinct types of receptors sited in the cell membranes of the various cell types present in the skin. The human kallikrein family comprises a large group of 15 serine proteases synthesized and secreted by different types of epithelial cells throughout the body, including the skin. At this site, they initiate a proteolytic cascade that generates the active forms of the proteases, some of which regulate skin desquamation, activation of cytokines, and antimicrobial peptides. Kinin peptides are formed by the action of plasma and tissue kallikreins on kininogens, two plasma proteins produced in the liver and other organs. Although kinins are well known for their proinflammatory abilities, in the skin they are also considered important modulators of keratinocyte differentiation. In this review, we summarize the contributions of the kallikreins and kallikrein-related peptidases family and those of kinins and their receptors in skin homeostasis, with special emphasis on their pathophysiological role.

Sex-specific Regulation of Prolactin Secretion by Pituitary Bradykinin Receptors.

Sex differences in the control of prolactin secretion are well documented. Sex-related differences in intrapituitary factors regulating lactotroph function have recently attracted attention. Sex differences in prolactinoma development are well documented in clinic, prolactinomas being more frequent in women but more aggressive in men, for poorly understood reasons. Kallikrein, the enzyme releasing kinins has been found in the pituitary, but there is no information on pituitary kinin receptors and their function. In the present work, we characterized pituitary bradykinin receptors (BRs) at the messenger RNA and protein levels in 2 mouse models of prolactinoma, Drd2 receptor gene inactivation and hCGß gene overexpression, in both males and females, wild type or genomically altered. BR B2 (B2R) accounted for 97% or more of total pituitary BRs in both models, regardless of genotype, and was present in lactotrophs, somatotrophs, and gonadotrophs. Male pituitaries displayed higher level of B2R than females, regardless of genotype. Pituitary B2R gene expression was downregulated by estrogen in both males and females but only in females by dopamine. Activation of B1R or B2R by selective pharmacological agonists induced prolactin release in male pituitaries but inhibited prolactin secretion in female pituitaries. Increased B2R content was observed in pituitaries of mutated animals developing prolactinomas, compared to their respective wild-type controls. The present study documents a novel sex-related difference in the control of prolactin secretion and suggests that kinins are involved, through B2R activation, in lactotroph function and prolactinoma development.

Kinin B1 receptor deficiency protects mice fed by cafeteria diet from abnormal glucose homeostasis.

The kallikrein-kinin system has been implicated in body weight and glucose homeostasis. Their major effectors act by binding to the kinin B2 and B1 receptors. It was assessed the role of the kinin B1 receptor in weight and glucose homeostasis in B1 receptor knockout mice (B1RKO) subjected to a cafeteria diet (CAF). Wild-type (WT) and B1RKO male mice (C57BL/6 background; 8 weeks old) were fed a standard diet (SD) or CAF for 14 weeks, ad libitum, and four groups were formed: WT-SD; B1RKO-SD; WT-CAF; B1RKO-CAF. Body weight and food intake were assessed weekly. It was performed glucose tolerance (GTT) and insulin tolerance tests (ITT), and HOMA-IR, HOMA-ß and HOMA-ß* 1/HOMA-IR were calculated. Islets from WT and B1RKO were isolated in order to measure the insulin secretion. Western blot was used to assess the hepatic AKT phosphorylation and qPCR to assess gene expression. CAF induced a higher body mass gain in B1RKO compared to WT mice. CAF diet increased epididymal fat depot mass, hepatic fat infiltration and hepatic AKT phosphorylation in both genotypes. However, B1RKO mice presented lower glycemic response during GTT when fed with CAF, and a lower glucose decrease in the ITT. This higher resistance was overcomed with higher insulin secretion when stimulated by high glucose, resulting in higher glucose uptake in the GTT when submitted to CAF, despite lower insulin sensitivity. Islets from B1RKO delivered 4 times more insulin in 3-month-old mice than islets from WT. The higher insulin disposition index and high insulin delivery of B1RKO can explain the decreased glucose excursion during GTT. In conclusion, CAF increased the ß-cell function in B1RKO mice, compensated by the diet-induced insulin resistance and resulting in a healthier glycemic response despite the higher weight gain.

Renal fibrosis due to multiple cisplatin treatment is exacerbated by kinin B1 receptor antagonism.

Cisplatin is a widely used chemotherapeutic drug, but its side effects are a major limiting factor. Nephrotoxicity occurs in one third of patients undergoing cisplatin treatment. The acute tubular injury caused by cisplatin often leads to a defective repair process, which translates into chronic renal disorders. In this way, cisplatin affects tubular cells, and maladaptive tubules regeneration will ultimately result in tubulointerstitial fibrosis. Kinins are well known for being important peptides in the regulation of inflammatory stimuli, and kinin B1 receptor deficiency and antagonism have been shown to be beneficial against acute cisplatin nephrotoxicity. This study aimed to analyze the effects of kinin B1 receptor deletion and antagonism against repeated cisplatin-induced chronic renal dysfunction and fibrosis. Both the deletion and the antagonism of B1 receptor exacerbated cisplatin-induced chronic renal dysfunction. Moreover, the inhibition of B1 receptor increased tubular injury and tubulointerstitial fibrosis after repeated treatment with cisplatin. The balance between M1/M2 macrophage polarization plays an important role in renal fibrosis. Kinin B1 receptor antagonism had no impact on M1 markers when compared to cisplatin. However, YM1, an M2 marker and an important molecule for the wound healing process, was decreased in mice treated with kinin B1 receptor antagonist, compared to cisplatin alone. Endothelin-1 levels were also increased in mice with B1 receptor inhibition. This study showed that kinin B1 receptor inhibition exacerbated cisplatin-induced chronic renal dysfunction and fibrosis, associated with reduced YM1 M2 marker expression, thus possibly affecting the wound healing process.

Role of Matricellular CCN Proteins in Skeletal Muscle: Focus on CCN2/CTGF and Its Regulation by Vasoactive Peptides.

The Cellular Communication Network (CCN) family of matricellular proteins comprises six proteins that share conserved structural features and play numerous biological roles. These proteins can interact with several receptors or soluble proteins, regulating cell signaling pathways in various tissues under physiological and pathological conditions. In the skeletal muscle of mammals, most of the six CCN family members are expressed during embryonic development or in adulthood. Their roles during the adult stage are related to the regulation of muscle mass and regeneration, maintaining vascularization, and the modulation of skeletal muscle fibrosis. This work reviews the CCNs proteins' role in skeletal muscle physiology and disease, focusing on skeletal muscle fibrosis and its regulation by Connective Tissue Growth factor (CCN2/CTGF). Furthermore, we review evidence on the modulation of fibrosis and CCN2/CTGF by the renin-angiotensin system and the kallikrein-kinin system of vasoactive peptides.

Intermittent hypoxia changes the interaction of the kinin-VEGF system and impairs myocardial angiogenesis in the hypertrophic heart.

Intermittent hypoxia (IH) is a feature of obstructive sleep apnea (OSA), a condition highly associated with hypertension-related cardiovascular diseases. Repeated episodes of IH contribute to imbalance of angiogenic growth factors in the hypertrophic heart, which is key in the progression of cardiovascular complications. In particular, the interaction between vascular endothelial growth factor (VEGF) and the kallikrein-kinin system (KKS) is essential for promoting angiogenesis. However, researchers have yet to investigate experimental models of IH that reproduce OSA, myocardial angiogenesis, and expression of KKS components. We examined temporal changes in cardiac angiogenesis in a mouse IH model. Adult male C57BI/6 J mice were implanted with Matrigel plugs and subjected to IH for 1-5 weeks with subsequent weekly histological evaluation of vascularization. Expression of VEGF and KKS components was also evaluated. After 3 weeks, in vivo myocardial angiogenesis and capillary density were decreased, accompanied by a late increase of VEGF and its type 2 receptor. Furthermore, IH increased left ventricular myocardium expression of the B2 bradykinin receptor, while reducing mRNA levels of B1 receptor. These results suggest that in IH, an unexpected response of the VEGF and KKS systems could explain the reduced capillary density and impaired angiogenesis in the hypoxic heart, with potential implications in hypertrophic heart malfunction.

Mesenchymal stem cell-glioblastoma interactions mediated via kinin receptors unveiled by cytometry.

Glioblastoma (GBM) is one of the most malignant and devastating brain tumors. The presence of highly therapy-resistant GBM cell subpopulations within the tumor mass, rapid invasion into brain tissues and reciprocal interactions with stromal cells in the tumor microenvironment contributes to an inevitable fatal prognosis for the patients. We highlight the most recent evidence of GBM cell crosstalk with mesenchymal stem cells (MSCs), which occurs either by direct cell-cell interactions via gap junctions and microtubules or cell fusion. MSCs and GBM paracrine interactions are commonly observed and involve cytokine signaling, regulating MSC tropism toward GBM, their intra-tumoral distribution, and immune system responses. MSC-promoted effects depending on their cytokine and receptor expression patterns are considered critical for GBM progression. MSC origin, tumor heterogeneity and plasticity may also determine the outcome of such interactions. Kinins and kinin-B1 and -B2 receptors play important roles in information flow between MSCs and GBM cells. Kinin-B1 receptor activity favors tumor migration and fusion of MSCs and GBM cells. Flow and image (tissue) cytometry are powerful tools to investigate GBM cell and MSC crosstalk and are applied to analyze and characterize several other cancer types.

The role of kallikrein-kinin and renin-angiotensin systems in COVID-19 infection.

In November 2019 the first cases of a novel acute respiratory syndrome has been reported in Wuhan province, China. Soon after, in January 2020 the World Health Organization declared a pandemic state due to the dissemination of a virus named SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2), the cause of coronavirus disease 2019 (COVID-19). Being an unknown disease, it is essential to assess not only its main characteristic features and overall clinical symptomatology but also its patient infection mode and propagation to design appropriate clinical interventions and treatments. In this review the pathophysiology of SARS-CoV-2 infection and how the virus enters the cells and activates the immune system are described. The role of three systems involved in the SARS- CoV-2 infection (renin-angiotensin, kinin and coagulation systems) is discussed with the objectives to identify and try to explain several of the events observed during the evolution of the disease and to suggest possible targets for therapeutic interventions.

Kinins and nitric oxide in patients with chronic chagas disease and systemic arterial hypertension.

BACKGROUND: Chronic Chagas disease (CCHD) associated with Systemic Arterial Hypertension (SAH) is frequently found in areas where the disease is endemic. The pathogenesis of patients with both pathologies (CCHD-SAH) is unsettled. Nitric Oxide (NO) and Kinins are important players in the myocardial inflammation process in experimental CCHD. No previous study has addressed this question in patients with CCHD, particularly in those with CCHD-SAH. Accordingly, this study was undertaken in an attempt to contribute to the understanding of the pathogenesis of patients with CCHD-SAH. METHODS: Thirty-seven patients with a positive serology for Chagas disease were enrolled; 15 had CCHD alone, 22 had CCHD-SAH (abnormal ECG/Doppler echocardiogram plus a systolic blood pressure > 140 mmHg or diastolic blood pressure > 90 mmHg on admission), and 11 had SAH alone. Thirty healthy individuals matched by age and sex served as controls. Plasma High-molecular (Hkg) and low-molecular weight (LKg) kininogens, plasma kallikrein levels (Pkal and Tcal), Kininase II, and plasma NO were measured. RESULTS: HKg and LKg were lower in CCHD-SAH patients in comparison with other groups (P < .0001). Pkal and Tcal were higher in CCHD-SAH patients in comparison with the other groups (P< .0001). Kininase II levels were similar in SAH, CCHD, and CCHD-SAH patients, but lower in comparison with controls (P< .0001). NO levels were similar in CCHD and CCHD-SAH patients, but higher in comparison with SAH patients and controls (P > .0001). CONCLUSION: Such findings suggest increased Kinin and NO activity in patients with CCHD-SAH, thus contributing to the understanding of the pathogenesis of this condition.

Kinin B1 Receptor Signaling in Skin Homeostasis and Wound Healing.

Kinins are proinflammatory peptides that are formed in the skin by the enzymatic action of tissue kallikrein (KLK1) on kininogens. Tissue kallikrein is produced by eccrine sweat glands and also by cells of the stratum granulosum and other skin appendages. Kinin formation may be favored during inflammatory skin disorders when plasma constituents, including kininogens, extravasate from venules and capillaries, which have increased permeability in response to the plethora of inflammatory mediators generated in the course of acute inflammation. By activating either kinin B1 or B2 receptors, kinins modulate keratinocyte differentiation, which relays on activation of several signaling systems that follows receptor stimulation. Participation of the kinin B1 receptor in wound healing is still a matter of controversy though some studies indicate that B1 receptor stimulation regulates keratinocyte migration by controlling metalloproteases 2 and 9 production and by improving wound closure in a mouse model. Development of more stable kinin B1 receptor agonists may be beneficial to modulate wound healing, especially if we take into account that the B1 receptor is up-regulated by inflammation and by cytokines generated in the inflamed microenvironment.

B1 and B2 kinin receptor blockade improves psoriasis-like disease.

BACKGROUND AND PURPOSE: The entire kallikrein-kinin system is present in the skin, and it is thought to exert a relevant role in cutaneous diseases, including psoriasis. The present study was designed to evaluate the relevance of kinin receptors in the development and progression of a model of psoriasis in mice. EXPERIMENTAL APPROACH: The effects of kinin B1 and B2 receptor knockout and of kinin receptor antagonists (SSR240612C or FR173657) were assessed in a model of psoriasis induced by imiquimod in C57BL/6 mice. Severity of psoriasis was assessed by histological and immunohistochemical assays of skin, along with objective scores based on the clinical psoriasis area and severity index. KEY RESULTS: Both kinin receptors were up-regulated following 6 days of imiquimod treatment. Kinin B1 and B2 receptor deficiency and the use of selective antagonists show morphological and histological improvement of the psoriasis hallmarks. This protective effect was associated with a decrease in undifferentiated and proliferating keratinocytes, decreased cellularity (neutrophils, macrophages, and CD4+ T lymphocytes), reduced γδ T cells, and lower accumulation of IL-17. The lack of B2 receptors resulted in reduced CD8+ T cells in the psoriatic skin. Relevantly, blocking kinin receptors reflected the improvement of psoriasis disease in the well-being behaviour of the mice. CONCLUSIONS AND IMPLICATIONS: Kinins exerted critical roles in imiquimod-induced psoriasis. Both B1 and B2 kinin receptors exacerbated the disease, influencing keratinocyte proliferation and immunopathology. Antagonists of one or even both kinin receptors might constitute a new strategy for the clinical treatment of psoriasis.

Activation of Toll-like receptor 2 induces B1 and B2 kinin receptors in human gingival fibroblasts and in mouse gingiva.

The regulation of the kallikrein-kinin system is an important mechanism controlling vasodilation and promoting inflammation. We aimed to investigate the role of Toll-like receptor 2 (TLR2) in regulating kinin B1 and B2 receptor expression in human gingival fibroblasts and in mouse gingiva. Both P. gingivalis LPS and the synthetic TLR2 agonist Pam2CSK4 increased kinin receptor transcripts. Silencing of TLR2, but not of TLR4, inhibited the induction of kinin receptor transcripts by both P. gingivalis LPS and Pam2CSK4. Human gingival fibroblasts (HGF) exposed to Pam2CSK4 increased binding sites for bradykinin (BK, B2 receptor agonist) and des-Arg10-Lys-bradykinin (DALBK, B1 receptor agonist). Pre-treatment of HGF for 24 h with Pam2CSK4 resulted in increased PGE2 release in response to BK and DALBK. The increase of B1 and B2 receptor transcripts by P. gingivalis LPS was not blocked by IL-1ß neutralizing antibody; TNF-α blocking antibody did not affect B1 receptor up-regulation, but partially blocked increase of B2 receptor mRNA. Injection of P. gingivalis LPS in mouse gingiva induced an increase of B1 and B2 receptor mRNA. These data show that activation of TLR2 in human gingival fibroblasts as well as in mouse gingival tissue leads to increase of B1 and B2 receptor mRNA and protein.

The role of kinins in the proliferation of fibroblast primed with TNF in scratch wound assay: Kinins and cell proliferation.

The aim of this study was to evaluate the involvement of both B1 and B2 kinins receptors (B1R and B2R) in the fibroblast proliferation induced by the cytokine tumour necrosis factor (TNF) attempting to establish an in vitro model of wound healing. Murine fibroblasts L-929 were cultivated in 24 wells plaque until total confluence (DMEM (Vitrocell®); 5% fetal bovine serum, 5% CO2, 37 °C) and then submitted to the scratch assay. The cells were treated with PBS, TNF (2 ng/mL) and/or mr-TNF antibody (200 µg/mL), or PDTC. The cells received the second set of treatment (3 h later): PBS; 1 µM HOE-140; 1 µM des-Arg9-Leu8-BK (DALBK) or 100 µM PDTC. TNF was able to increase the cell proliferation when compared with the group treated with PBS. The co-treatment with the TNF antibody completely reversed the TNF effect. The TNF-proliferative effect was blocked by B1 (DALBK) and B2 (HOE-140) kinin receptor antagonists administered separately or along, suggesting the involvement of both receptors in the TNF mechanism of action. Furthermore, the treatment with a NF-ĸB inhibitor PDTC completely blocked the cell proliferation. The TNF cell proliferation was incremented with BK (1 µM) treatment, and its effect was totally reversed by HOE-140 treatment. No effect was observed for TNF plus DABK. Eventually, TNF treatment was able to increase TNF level in the growing medium; however, this increase was suppressed by BK treatment. These results suggest that TNF induces cell proliferation and the induced signalling cascade has the B2R participation. All these events seem to be totally dependent on the NF-ĸB activation. These inflammatory mediators can improve the wound healing in the resolution of inflammation.

Subverting bradykinin-evoked inflammation by co-opting the contact system: lessons from survival strategies of Trypanosoma cruzi.

PURPOSE OF REVIEW: During Chagas disease, Trypanosoma cruzi alternates between intracellular and extracellular developmental forms. After presenting an overview about the roles of the contact system in immunity, I will review experimental studies showing that activation of the kallikrein-kinin system (KKS) translates into mutual benefits to the host/parasite relationship. RECENT FINDINGS: T. cruzi trypomastigotes initiate inflammation by activating tissue-resident innate sentinel cells via the TLR2/CXCR2 pathway. Following neutrophil-evoked microvascular leakage, the parasite's major cysteine protease (cruzipain) cleaves plasma-borne kininogens and complement C5. Tightly regulated by angiotensin-converting enzyme (ACE), kinins and C5a in turn further propagate inflammation via iterative cycles of mast cell degranulation, contact system activation, bradykinin release and activation of endothelial bradykinin B2 receptors (B2R). Recently, studies in the intracardiac model of infection revealed a dichotomic role for bradykinin and endothelin-1: generated upon contact activation (mast cell/KKS pathway), these pro-oedematogenic peptides reciprocally stimulate trypomastigote invasion of heart cells that naturally overexpress B2R and endothelin receptors (ETaR/ETbR). SUMMARY: Studies focusing on the immunopathogenesis of Chagas disease revealed that the contact system plays a dual role in host/parasite balance: T. cruzi co-opts bradykinin-induced plasma leakage as a strategy to increment heart parasitism and increase immune resistance by upregulating type-1 effector T-cell production in secondary lymphoid tissues.

Functional interrelationships between the kallikrein-related peptidases family and the classical kinin system in the human neutrophil.

In the human neutrophil, kallikrein-related peptidases (KLKs) have a significant functional relationship with the classical kinin system as a kinin B1 receptor agonist induces secretion of KLK1, KLK6, KLK10, KLK13 and KLK14 into the medium. Secretion of KLK1, the kinin-forming enzyme, may perpetuate formation of kinin in the inflammatory milieu by hydrolyzing extravasated kininogens present in tissue edema. Secretion of KLKs into the inflammatory milieu, induced by kinins or other proinflammatory mediators, provides the human neutrophil with a wide range of molecular interactions to hydrolyze different cellular and extracellular matrix components, which may be of critical relevance in different mechanisms involving inflammation.

Kn-Ba: a novel serine protease isolated from Bitis arietans snake venom with fibrinogenolytic and kinin-releasing activities

Background: Bitis arietans is a venomous snake found in sub-Saharan Africa and in parts of Morocco and Saudi Arabia. The envenomation is characterized by local and systemic reactions including pain, blistering, edema and tissue damage, besides hemostatic and cardiovascular disturbances, which can cause death or permanent disabilities in its victims. However, the action mechanisms that provoke these effects remain poorly understood, especially the activities of purified venom components. Therefore, in order to elucidate the molecular mechanisms that make the Bitis arietans venom so potent and harmful to human beings, this study reports the isolation and biochemical characterization of a snake venom serine protease (SVSP). Methods: Solubilized venom was fractionated by molecular exclusion chromatography and the proteolytic activity was determined using fluorescent substrates. The peaks that showed serine protease activity were determined by blocking the proteolytic activity with site-directed inhibitors. In sequence, the fraction of interest was submitted to another cycle of molecular exclusion chromatography. The purified serine protease was identified by mass spectrometry and characterized biochemically and immunochemically. Results: A serine protease of 33 kDa with fibrinogen-degrading and kinin-releasing activities was isolated, described, and designated herein as Kn-Ba. The experimental Butantan Institute antivenom produced against Bitis arietans venom inhibited the Kn-Ba activity. Conclusions: The in vitro activities of Kn-Ba can be correlated with the capacity of the venom to provoke bleeding and clotting disorders as well as hypotension, which are common symptoms presented by envenomed victims. Obtaining satisfactory Kn-Ba inhibition through the experimental antivenom is important, given the WHO's recommendation of immunotherapy in cases of human accidents with venomous snakes.(AU)

Papel do receptor B2 de cininas na terapia da neurodegeneração dopaminérgica em modelo animal / Targeting Kinin-B2 receptors for the treatment of dopaminergic neurodegeneration in an animal mode

A Doença de Parkinson (DP) é um distúrbio neurodegenerativo, caracterizada em parte pela perda de neurônios dopaminérgicos da via nigroestriatal, originada na substância negra com projeções para o estriado, causando vários déficits motores. Atualmente, o tratamento mais utilizado é a administração de L-DOPA, um análogo da dopamina. Porém, essa droga apresenta eficácia limitada e induz diversos efeitos colaterais. A exploração dos efeitos neuroprotetores, proliferativos e neuroregenerativos da bradicinina (BK) em modelo animal de DP pode conduzir à substituição celular do tecido lesionado pela 6-hidroxidopamina (6-OHDA). De fato, a BK e seus receptores possuem um grande espectro de ações fisiológicas, estando classicamente envolvida no controle da homeostase cardiovascular e inflamação, além de exercer efeitos protetores em fisiopatologias do sistema nervoso, como em modelos de acidente vascular cerebral. Vários tipos celulares têm suas vias de sinalização associadas à ativação do receptor B2 de cininas (B2BKR). Trabalhos anteriores de nosso grupo mostraram que a BK está envolvida na diferenciação neural de células progenitoras neurais por um loop autócrino que resulta em ativação do B2BKR. Os resultados apresentados neste trabalho mostram a eficácia do tratamento com BK, um agonista de B2BKR, em animais submetidos à lesão da via nigro-estriatal induzida por 6-OHDA. Além disso, há uma recuperação comportamental e histológica desses animais quando tratados com Captopril®, um potencializador dos efeitos farmacológicos da BK, e com [Phe8Ψ(CH-NH)Arg9]-Bradicinina, agonista estável do receptor B2BKR. Assim, concluímos que a ativação de B2BKR pela BK desencadeiaum processo de neuroregeneração dopaminérgica de animais submetidos à lesão por 6-OHDA. Trabalhos recentes mostram que o receptor B2BKR desempenha um importante papel neuroprotetor em modelo animal da Doença de Alzheimer, o que corrobora nossos achados. Juntos, esses resultados contribuem para o estabelecimento da ação neuroprotetora e neurorregenerativa da BK no modelo de animal de neurodegeneração dopaminérgica, tornando-a uma excelente candidata para aplicação em terapias de reparo neuronal

Parkinson's disease (PD) is a neurodegenerative disorder partially characterized by the loss of dopaminergic neurons from the nigrostriatal pathway, originated in the substantia nigra with projections to the striatum, which causes several motor deficits. Currently, the most commonly used drug for PD treatment is levodopa. However, it has limited efficacy and induces several side effects. Elucidation of the neuroprotective, proliferative and neuroregenerative effects of bradykinin (BK) in animal models of PD can culminate in cellular replacement of the tissue damaged by 6-hydroxydopamine (6-OHDA). In fact, BK and its receptor have several physiological effects, being classically involved in the control of cardiovascular homeostasis and inflammation. Besides, BK exerts protective effects on nervous system pathophysiology, as observed in stroke models. Several cell types have their signaling pathways associated with the B2 kinin receptor (B2BKR) activation. Previous work from our group showed that BK is involved in differentiation of neural progenitor cells by an autocrine loop that results in activation of B2BKR. The results presented in this thesis show the efficacy of treatment with BK, through B2BKR activation, in animals submitted to nigrostriatal pathway injury induced by 6-OH dopamine. Furthermore, behavioral and histological recoveries of these animals were observed when treated with Captopril®, a potentiator of BK pharmacological effects, and with [Phe8Ψ (CH-NH) Arg9] -BK, a stable agonist of the B2BKR receptor. Thus, we conclude that BK activation of B2BKR triggers neuroregenerative processes in animals submitted to 6- OHDA injury. Recent studies showed that the B2BKR receptor plays an important neuroprotective role in an animal model of Alzheimer's disease, which corroboratesour findings. Together, these results contribute to the establishment of the neuroprotective and neuroregenerative actions of BK - an excellent candidate for neural repair therapies

Kinin B2-Receptor in human iPSC differentiation into cardiomyocytes / Receptor B2 de cininas na diferenciação de iPSC humanas em cardiomiócitos

Cardiovascular diseases are responsible for almost one third of all global deaths yearly, and therefore are largely studied. Cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CM) have emerged as an exciting technology for cardiac disease modelling and personalised therapy. Nevertheless, issues concerning functional and molecular maturation are still faced. In addition to this, differentiation protocols generally yield a heterogeneous mixed population comprised of nodal, atrial and ventricular-like subtypes, being unsuitable for therapeutic purposes. Bradykinin (BK) is a vasoactive peptide which exerts important physiological roles in the cardiovascular system, having been previously described as important for cellular, keratinocyte and skeletal muscle differentiation. This project performed in cooperation with PluriCell Biotech, a startup specialized in the production and differentiation of hiPSC-CM, has sought (1) characterizing gene and protein expression of molecular markers of maturation and of subtype specification throughout of differentiation; (2) Assessing the electrical functionality of hiPSC-CM through the characterization of subtype-specific action potentials (APs) and (3) Investigating whether the progress of hiPSCCM maturation is regulated by BK through kinin-B2 receptors (B2R). Our results have validated the model that proposes a developmental-dependent switch between skeletal (ssTnI) and cardiac (cTnI) isoforms of troponin I as differentiation progresses, at least to some extent. Furthermore, prolonged time in culture has resulted in higher levels of expression of the ventricular marker MLC2v and in increased rates of ventricular-like action APs. Electrophysiological analysis of hiPSC-CM reveals a mixed population with AP morphologies correspondent to nodal, atrial and ventricular subtypes, all showing pronounced automaticity as well as other features of immature cardiomyocytes, such as low amplitude and depolarization velocity. Such findings are coherent with those from other groups who have attempted to differentiate mature native-like cardiac cells from pluripotent stem cells sources, without fully succeeding. After showing that differentiating hiPSC-CM express a functional and responsive B2R, the receptor was subjected to chronic activation with 10µM BK and 1µM BK or inhibition with 5µM Firazyr+BK. Even though B2R modulation has not interfered negatively with differentiation yields nor cell morphology, analysis of gene andprotein expression of ssTnI or cTnI and of the ventricular marker MLC2v, have revealed no significant results in comparison to untreated controls. This suggests that BK does not interfere on hiPSC-CM maturation nor subtype specification, although we cannot rule out that it could be leading to other unexplored effects. We recommend a closer look into which intracellular signalling pathways become active upon B2R stimulation in hiPSC-CM, in order to narrow down cellular processes for further investigation

Doenças cardiovasculares são responsáveis por quase um terço de todas as mortes globais anualmente, e por isto o sistema cardiovascular é amplamente estudado. Cardiomiócitos derivados a partir de células-tronco pluripotentes induzidas humanas (hiPSCCM) emergiram como uma promissora tecnologia para modelagem de doenças cardíacas e terapia personalizada. No entanto, desafios acerca de sua maturação funcional e molecular ainda são enfrentados. Além disso, protocolos de diferenciação geralmente levam à obtenção de populações heterogêneas contendo células com fenótipos similares aos de cardiomiócitos nodais, atriais e ventriculares sendo, portanto, inapropriadas para fins terapêuticos. A bradicinina (BK) é um peptídio vasoativo que exerce importantes papeis fisiológicos no sistema cardiovascular, além de ter sido previamente descrita como importante para a diferenciação neuronal, de queratinócitos e de músculo esquelético. Este projeto foi realizado em colaboração com a empresa PluriCell Biotech, uma startup especializada na produção e diferenciação de hiPSC-CM, e buscou (1) caracterizar a expressão gênica e proteíca de marcadores moleculares de maturação e de especificação de subtipos cardíacos durante a diferenciação; (2) avaliar a funcionalidade elétrica de hiPSC-CM por meio da caracterização de seus potenciais de ação (PAs) e (3) Investigar se o progresso da diferenciação de hiPSCCM é regulado por bradicinina por meio do receptor B2 (B2R). Nossos resultados validaram o modelo que propõe um switch na expressão das isoformas funcionais de troponina I esquelética (ssTnI) e cardíaca (cTnI), durante o desenvolvimento e diferenciação celular, pelo menos parcialmente. Além disso, tempo prolongado em cultura resultou em maiores níveis de expressão do marcador ventricular MLC2v, assim como maiores frequências de PAs com morfologias similares a de cardiomiócitos ventriculares. Análise eletrofisiológica de hiPSCCM revelam a existência de uma população mista contendo PAs correspondentes aos subtipos nodais, atriais e ventriculares, assim como pronunciada automaticidade e outros atributos típicos de cardiomiócitos imaturos, como baixa amplitude e devagar velocidade de despolarização. Estes resultados são coerentes com os de outros grupos que ainda não foram totalmente bem-sucedidos em diferenciar células cardíacas maduras similares acardiomiócitos nativos a partir de células-troncos pluripotentes. Após mostrar que as hiPSCCM expressam receptores B2 funcionais e responsivos, submetemos o receptor a uma ativação crônica com BK 10µM e BK 1µM ou inibição crônica com Firazyr 5µM + BK. Apesar da modulação do B2R não ter interferido de forma negativa no rendimento da diferenciação ou na morfologia celular, análise de expressão gênica e proteica de ssTnI e cTnI e do marcador ventricular MLC2v não revelou resultados significativos em comparação aos controles não-tratados. Isto sugere que a BK não interfere na maturação e especificação de subtipos cardíacos em hiPSC-CM, apesar de não podermos ignorar o fato de que ela poderia estar desencadeando outros efeitos inexplorados. Nós recomendamos um estudo mais aprofundado acerca de quais vias de sinalização se tornam ativas após estimulação do receptor B2 em hiPSC-CM, com o objetivo de afunilar quais processos celulares poderiam ser investigados em uma próxima etapa deste estudo

Kn-Ba: a novel serine protease isolated from Bitis arietans snake venom with fibrinogenolytic and kinin-releasing activities

Bitis arietans is a venomous snake found in sub-Saharan Africa and in parts of Morocco and Saudi Arabia. The envenomation is characterized by local and systemic reactions including pain, blistering, edema and tissue damage, besides hemostatic and cardiovascular disturbances, which can cause death or permanent disabilities in its victims. However, the action mechanisms that provoke these effects remain poorly understood, especially the activities of purified venom components. Therefore, in order to elucidate the molecular mechanisms that make the Bitis arietans venom so potent and harmful to human beings, this study reports the isolation and biochemical characterization of a snake venom serine protease (SVSP). Methods: Solubilized venom was fractionated by molecular exclusion chromatography and the proteolytic activity was determined using fluorescent substrates. The peaks that showed serine protease activity were determined by blocking the proteolytic activity with site-directed inhibitors. In sequence, the fraction of interest was submitted to another cycle of molecular exclusion chromatography. The purified serine protease was identified by mass spectrometry and characterized biochemically and immunochemically. Results: A serine protease of 33 kDa with fibrinogen-degrading and kinin-releasing activities was isolated, described, and designated herein as Kn-Ba. The experimental Butantan Institute antivenom produced against Bitis arietans venom inhibited the Kn-Ba activity. Conclusions: The in vitro activities of Kn-Ba can be correlated with the capacity of the venom to provoke bleeding and clotting disorders as well as hypotension, which are common symptoms presented by envenomed victims. Obtaining satisfactory Kn-Ba inhibition through the experimental antivenom is important, given the WHO's recommendation of immunotherapy in cases of human accidents with venomous snakes.(AU)