RESUMEN

BACKGROUND AND PURPOSE: Ulinastatin has beneficial effects in patients undergoing coronary artery bypass grafting (CABG) surgery due to its anti-inflammatory properties, but the underlying mechanism remains unclear. EXPERIMENTAL APPROACH: We used samples from patients undergoing CABG, a model of cardiac ischaemia-reperfusion injury (IRI) in mice and murine cardiac endothelial cell cultures to investigate links between ulinastatin, the kallikrein-kinin system (KKS), endothelial dysfunction and cardiac inflammation in the response to ischaemia/reperfusion injury (IRI). These links were assessed using clinical investigations, in vitro and in vivo experiments and RNA sequencing analysis. KEY RESULTS: Ulinastatin inhibited the activity of tissue kallikrein, a key enzyme of the KKS, at 24 h after CABG surgery, which was verified in our murine cardiac ischaemia-reperfusion model. Under normal conditions, ulinastatin only inhibited kallikrein activity but did not affect bradykinin (B1/B2) receptors. Ulinastatin protected against IRI, in vivo and in vitro, by suppressing activation of the kallikrein-kinin system and down-regulating B1/B2 receptor-related signalling pathways including ERK/ iNOS, which resulted in enhanced endothelial barrier function, mitigation of inflammation and oedema, decreased infarct size, improved cardiac function and decreased mortality. Inhibition of kallikrein and knockdown of B1, but not B2 receptors prevented ERK translocation into the nucleus, reducing reperfusion-induced injury in murine cardiac endothelial cells. CONCLUSIONS AND IMPLICATIONS: Treatment with ulinastatin exerts a protective influence on cardiac reperfusion by suppressing activation of the kallikrein-kinin system. Our findings highlight the potential of targeting kallikrein /bradykinin receptors to alleviate endothelial dysfunction, thus improving cardiac IRI.

RESUMEN

Epigenetic changes have long-lasting impacts, which influence the epigenome and are maintained during cell division. Thus, human genome changes have required a very long timescale to become a major contributor to the current obesity pandemic. Whereas bidirectional effects of coronavirus disease 2019 (COVID-19) and obesity pandemics have given the opportunity to explore, how the viral microribonucleic acids (miRNAs) use the human's transcriptional machinery that regulate gene expression at a posttranscriptional level. Obesity and its related comorbidity, type 2 diabetes (T2D), and new-onset diabetes due to severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) are additional risk factors, which increase the severity of COVID-19 and its related mortality. The higher mortality rate of these patients is dependent on severe cytokine storm, which is the sum of the additional cytokine production by concomitant comorbidities and own cytokine synthesis of COVID-19. Patients with obesity facilitate the SARS-CoV-2 entry to host cell via increasing the host's cell receptor expression and modifying the host cell proteases. After entering the host cells, the SARS-CoV-2 genome directly functions as a messenger ribonucleic acid (mRNA) and encodes a set of nonstructural proteins via processing by the own proteases, main protease (Mpro), and papain-like protease (PLpro) to initiate viral genome replication and transcription. Following viral invasion, SARS-CoV-2 infection reduces insulin secretion via either inducing ß-cell apoptosis or reducing intensity of angiotensin-converting enzyme 2 (ACE2) receptors and leads to new-onset diabetes. Since both T2D and severity of COVID-19 are associated with the increased serum levels of pro-inflammatory cytokines, high glucose levels in T2D aggravate SARS-CoV-2 infection. Elevated neopterin (NPT) value due to persistent interferon gamma (IFN-γ)-mediated monocyte-macrophage activation is an indicator of hyperactivated pro-inflammatory phenotype M1 macrophages. Thus, NPT could be a reliable biomarker for the simultaneously occurring COVID-19-, obesity- and T2D-induced cytokine storm. While host miRNAs attack viral RNAs, viral miRNAs target host transcripts. Eventually, the expression rate and type of miRNAs also are different in COVID-19 patients with different viral loads. It is concluded that specific miRNA signatures in macrophage activation phase may provide an opportunity to become aware of the severity of COVID-19 in patients with obesity and obesity-related T2D.

Asunto(s)

COVID-19 , Síndrome de Activación Macrofágica , Obesidad , SARS-CoV-2 , Humanos , COVID-19/virología , COVID-19/epidemiología , COVID-19/inmunología , COVID-19/complicaciones , Obesidad/complicaciones , Obesidad/metabolismo , Obesidad/epidemiología , Obesidad/virología , SARS-CoV-2/fisiología , SARS-CoV-2/patogenicidad , Síndrome de Activación Macrofágica/virología , Síndrome de Activación Macrofágica/epidemiología , Diabetes Mellitus Tipo 2/epidemiología , Diabetes Mellitus Tipo 2/virología , Diabetes Mellitus Tipo 2/metabolismo , Pandemias , MicroARNs/genética , MicroARNs/metabolismo , Citocinas/metabolismo , Síndrome de Liberación de Citoquinas/inmunología , Síndrome de Liberación de Citoquinas/virología

RESUMEN

Serine protease inhibitors (serpins) participate in the regulation of inflammation, blood coagulation, and complement activation in humans. This research aimed to identify and characterize such inhibitors of the human liver fluke Opisthorchis viverrini. Parasite proteins that might contribute to the modulation of host physiology are of particular interest, especially as chronic opisthorchiasis increases the risk of developing biliary cancer. BLAST was used to find hypothetical serpins predicted from the parasite genome data. RNA extraction and reverse transcriptase PCR were used to isolate a serpin cDNA and to determine developmental transcript abundance. The evolutionary relation to other trematode serpins was revealed by phylogenetic analysis. Recombinant serpin was expressed in Escherichia coli and used to test the immunoreactivity of human opisthorchiasis sera and the inhibition of human serine proteases. A substantial serpin family with high sequence divergence among the members was found in the genus Opisthorchis. A serpin, different from previously analyzed trematode serpins, was cloned. The transcript was only detected in metacercariae and newly excysted juveniles. Human opisthorchiasis sera showed statistically significant reactivity to recombinant serpin. The serpin caused moderate inhibition of thrombin and low inhibition of kallikrein and chymotrypsin. This parasite serpin could be further evaluated as a diagnostic tool for early infection. Kallikrein and thrombin are involved in fibrinolysis; therefore, further research should explore the effects of the parasite serpin on this process.

RESUMEN

Background: There are indications for sex-specific differences regarding the association between kallikrein-8 (KLK8) and cognitive impairment in early stages of Alzheimer's disease for which KLK8 may be an early blood-based biomarker. These may be due to different levels of sex hormones. To correctly interpret KLK8 blood concentrations, sex-specific analyses are needed. Objective: The aim of our exploratory study was to investigate sex-specific differences in blood-based KLK8 in participants of the population-based Heinz Nixdorf Recall study with different cognitive status and the association between KLK8 and sex hormones. Methods: In 290 participants (45% women, 69.7±7.4 years (mean±SD)) we investigated sex-specific serum KLK8 differences between cognitively unimpaired (CU, 43%) and cognitively impaired (CI) participants and the association between KLK8 and dehydroepiandrosteronsulfate (DHEAS), estradiol and testosterone, using adjusted multiple linear regression. Results: The mean±SD KLK8 was similar for CU men (808.1±729.6 pg/ml) and women (795.9±577.7 pg/ml); adjusted mean-difference [95%-CI]: -95.3 [-324.1;133.5] pg/ml. KLK8 was lower in CI women (783.5±498.7 pg/ml) than men (1048.4±829 pg/ml); -261 [-493.1; -29] pg/ml. In men but not women, there was a weak indication for a positive slope between estradiol (11.9 [-0.4;24.3] pg/ml) and DHEAS (1.4 [-0.5;3.3] pg/ml) with KLK8, while testosterone had no impact. Conclusions: The results suggested a different role for KLK8 in the development of cognitive impairment in men and women, potentially influenced by sex hormones. To use blood KLK8 as an early biomarker, further research on hormonal regulation of KLK8 expression is needed as a part of the investigation of the KLK8 involvement in cognitive impairment and Alzheimer's disease pathology.

Asunto(s)

Biomarcadores , Disfunción Cognitiva , Calicreínas , Humanos , Femenino , Masculino , Calicreínas/sangre , Anciano , Disfunción Cognitiva/sangre , Biomarcadores/sangre , Persona de Mediana Edad , Testosterona/sangre , Estradiol/sangre , Caracteres Sexuales , Sulfato de Deshidroepiandrosterona/sangre , Enfermedad de Alzheimer/sangre , Factores Sexuales

RESUMEN

Aprotinin is a broad-spectrum inhibitor of human proteases that has been approved for the treatment of bleeding in single coronary artery bypass surgery because of its potent antifibrinolytic actions. Following the outbreak of the COVID-19 pandemic, there was an urgent need to find new antiviral drugs. Aprotinin is a good candidate for therapeutic repositioning as a broad-spectrum antiviral drug and for treating the symptomatic processes that characterise viral respiratory diseases, including COVID-19. This is due to its strong pharmacological ability to inhibit a plethora of host proteases used by respiratory viruses in their infective mechanisms. The proteases allow the cleavage and conformational change of proteins that make up their viral capsid, and thus enable them to anchor themselves by recognition of their target in the epithelial cell. In addition, the activation of these proteases initiates the inflammatory process that triggers the infection. The attraction of the drug is not only its pharmacodynamic characteristics but also the possibility of administration by the inhalation route, avoiding unwanted systemic effects. This, together with the low cost of treatment (≈2 Euro/dose), makes it a good candidate to reach countries with lower economic means. In this article, we will discuss the pharmacodynamic, pharmacokinetic, and toxicological characteristics of aprotinin administered by the inhalation route; analyse the main advances in our knowledge of this medication; and the future directions that should be taken in research in order to reposition this medication in therapeutics.

Asunto(s)

Antivirales , Aprotinina , Tratamiento Farmacológico de COVID-19 , SARS-CoV-2 , Aprotinina/uso terapéutico , Aprotinina/farmacología , Aprotinina/química , Humanos , Antivirales/uso terapéutico , Antivirales/farmacología , Antivirales/administración & dosificación , Administración por Inhalación , SARS-CoV-2/efectos de los fármacos , COVID-19/virología , Animales , Reposicionamiento de Medicamentos/métodos , Inhibidores de Serina Proteinasa/uso terapéutico , Inhibidores de Serina Proteinasa/farmacología , Inhibidores de Serina Proteinasa/administración & dosificación

RESUMEN

Proteases are produced and released in the mucosal cells of the respiratory tract and have important physiological functions, for example, maintaining airway humidification to allow proper gas exchange. The infectious mechanism of severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2), which causes coronavirus disease 2019 (COVID-19), takes advantage of host proteases in two ways: to change the spatial conformation of the spike (S) protein via endoproteolysis (e.g., transmembrane serine protease type 2 (TMPRSS2)) and as a target to anchor to epithelial cells (e.g., angiotensin-converting enzyme 2 (ACE2)). This infectious process leads to an imbalance in the mucosa between the release and action of proteases versus regulation by anti-proteases, which contributes to the exacerbation of the inflammatory and prothrombotic response in COVID-19. In this article, we describe the most important proteases that are affected in COVID-19, and how their overactivation affects the three main physiological systems in which they participate: the complement system and the kinin-kallikrein system (KKS), which both form part of the contact system of innate immunity, and the renin-angiotensin-aldosterone system (RAAS). We aim to elucidate the pathophysiological bases of COVID-19 in the context of the imbalance between the action of proteases and anti-proteases to understand the mechanism of aprotinin action (a panprotease inhibitor). In a second-part review, titled "Aprotinin (II): Inhalational Administration for the Treatment of COVID-19 and Other Viral Conditions", we explain in depth the pharmacodynamics, pharmacokinetics, toxicity, and use of aprotinin as an antiviral drug.

Asunto(s)

Aprotinina , Tratamiento Farmacológico de COVID-19 , COVID-19 , SARS-CoV-2 , Humanos , Aprotinina/farmacología , Aprotinina/uso terapéutico , Aprotinina/metabolismo , SARS-CoV-2/efectos de los fármacos , COVID-19/virología , COVID-19/metabolismo , Enzima Convertidora de Angiotensina 2/metabolismo , Péptido Hidrolasas/metabolismo , Glicoproteína de la Espiga del Coronavirus/metabolismo , Serina Endopeptidasas/metabolismo

RESUMEN

Hereditary angioedema due to C1 inhibitor deficiency (HAE) is a rare inborn error of immunity that presents with episodic swelling. Management is multifaceted and includes on-demand treatment of swelling episodes, short-term prophylaxis to prevent swelling episodes from procedures, and long-term prophylaxis (LTP) to prevent angioedema on an ongoing basis. All approved on-demand therapies are parenteral, necessitating patient training for home administration, particularly intravenous C1 inhibitor. These complexities can result in care gaps for rural HAE patients. We conducted a cross-sectional study at our Angioedema Center of Reference and Excellence to assess the care provided to urban and rural patients. The proportion of patients receiving LTP, proportion of patients diagnosed as children, and disease control measured using the Angioedema Control Test (AECT) were collected. Logistic and Poisson regression models adjusted for age and sex were used to compare the two groups. The proportion using LTP was similar at 62% and 61% in urban and rural patients, respectively (odds ratio [OR] 1.01 (CI 95% 0.34-2.99)). Among urban patients, 52% were diagnosed as children compared to 60% among rural residents (1.43 (0.37-5.56)). The mean (IQR) AECT score was 14.0 (8.5-15.5) in urban patients and 13.0 (10.0-14.0) in rural patients (Poisson ß -0.001 (-0.23-0.23). These data indicate that rural patients received similar high-quality care. We attribute these findings to the centralized care model employed in which HAE patients in the region are seen at a single comprehensive care clinic.

Asunto(s)

Angioedemas Hereditarios , Población Rural , Humanos , Masculino , Femenino , Estudios Transversales , Angioedemas Hereditarios/diagnóstico , Angioedemas Hereditarios/terapia , Niño , Adulto , Adolescente , Preescolar , Población Urbana , Adulto Joven , Proteína Inhibidora del Complemento C1/uso terapéutico , Persona de Mediana Edad

Asunto(s)

Angioedemas Hereditarios , Edición Génica , Humanos , Angioedemas Hereditarios/genética , Angioedemas Hereditarios/tratamiento farmacológico , Femenino , Masculino , Adulto , Persona de Mediana Edad , Sistemas CRISPR-Cas

RESUMEN

(1) Background: This cross-sectional investigation appreciated the role of serum C-reactive protein (CRP), several hematologic-cell markers, and salivary inflammation-related molecules [calprotectin (S100A8/A9), interleukin-1ß (IL-1ß), kallikrein] to predict periodontitis in patients with atherosclerotic cardiovascular disease (ACVD), arrhythmia, or both. Also, we appreciated the relationship between the inflammatory burden and periodontal destruction with the type of cardiac pathology. (2) Methods: Demographic, behavioral characteristics, periodontal indicators, blood parameters, and saliva samples were collected. (3) Results: All 148 patients exhibited stage II or III/IV periodontitis. Stage III/IV cases exhibited significantly increased S100A8/A9 levels (p = 0.004). A positive correlation between S100A8/A9 and IL-1ß [0.35 (<0.001)], kallikrein [0.55 (<0.001)], and CRP [0.28 (<0.001)] was observed. Patients with complex cardiac involvement had a significantly higher number of sites with attachment loss ≥ 5 mm [19 (3-30)] compared to individuals with only arrhythmia [9 (3.25-18)] or ACVD [5 (1-12)] [0.048♦ {0.162/0.496/0.14}]. (4) Conclusions: Severe, extensive attachment loss may be indicative of patients with complex cardiac conditions, which underscores the essential role of periodontal status in relation to systemic diseases. The correlations between the rising trends of the inflammatory parameters suggest a potential interconnection between oral and systemic inflammation.

RESUMEN

Activation of the kallikrein-kinin system promotes vascular leakage, inflammation, and neurodegeneration in ischemic stroke. Inhibition of plasma kallikrein (PK) - a key component of the KKS - in the acute phase of ischemic stroke has been reported to reduce thrombosis, inflammation, and damage to the blood-brain barrier. However, the role of PK during the recovery phase after cerebral ischemia is unknown. To this end, we evaluated the effect of subacute PK inhibition starting from day 3 on the recovery process after transient middle artery occlusion (tMCAO). Our study demonstrated a protective effect of PK inhibition by reducing infarct volume and improving functional outcome at day 7 after tMCAO. In addition, we observed reduced thrombus formation in cerebral microvessels, fewer infiltrated immune cells, and an improvement in blood-brain barrier integrity. This protective effect was facilitated by promoting tight junction reintegration, reducing detrimental matrix metalloproteinases, and upregulating regenerative angiogenic markers. Our findings suggest that PK inhibition in the subacute phase might be a promising approach to accelerate the post-stroke recovery process.

Asunto(s)

Calicreína Plasmática , Recuperación de la Función , Animales , Recuperación de la Función/efectos de los fármacos , Recuperación de la Función/fisiología , Masculino , Calicreína Plasmática/antagonistas & inhibidores , Calicreína Plasmática/metabolismo , Ratones , Ratones Endogámicos C57BL , Infarto de la Arteria Cerebral Media , Barrera Hematoencefálica/efectos de los fármacos , Barrera Hematoencefálica/metabolismo , Accidente Cerebrovascular/tratamiento farmacológico , Trombosis , Accidente Cerebrovascular Isquémico/tratamiento farmacológico , Inflamación

RESUMEN

Ecallantide comprises Kunitz Domain 1 of Tissue Factor Pathway Inhibitor, mutated at seven amino acid positions to inhibit plasma kallikrein (PK). It is used to treat acute hereditary angioedema (HAE). We appended hexahistidine tags to the N- or C-terminus of recombinant Ecallantide (rEcall) and expressed and purified the resulting proteins, with or without fusion to human serum albumin (HSA), using Pichia pastoris. The inhibitory constant (Ki) of rEcall-H6 or H6-rEcall for PK was not increased by albumin fusion. When 125I-labelled rEcall proteins were injected intravenously into mice, the area under the clearance curve (AUC) was significantly increased, 3.4- and 3.6-fold, for fusion proteins H6-rEcall-HSA and HSA-rEcall-H6 versus their unfused counterparts but remained 2- to 3-fold less than that of HSA-H6. The terminal half-life of H6-rEcall-HSA and HSA-H6 did not differ, although that of HSA-rEcall-H6 was significantly shorter than either other protein. Receptor Associated Protein (RAP), a Low-density lipoprotein Receptor-related Protein (LRP1) antagonist, competed H6-rEcall-HSA clearance more effectively than intravenous immunoglobulin (IVIg), a neonatal Fc receptor (FcRn) antagonist. HSA fusion decreases rEcall clearance in vivo, but LRP1-mediated clearance remains more important than FcRn-mediated recycling for rEcall fusion proteins. The properties of H6-rEcall-HSA warrant investigation in a murine model of HAE.

Asunto(s)

Proteínas Recombinantes de Fusión , Animales , Proteínas Recombinantes de Fusión/farmacocinética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/farmacología , Proteínas Recombinantes de Fusión/química , Ratones , Humanos , Semivida , Calicreína Plasmática/metabolismo , Calicreína Plasmática/genética , Albúmina Sérica Humana/química , Albúmina Sérica Humana/genética , Albúmina Sérica Humana/metabolismo , Saccharomycetales/genética , Saccharomycetales/metabolismo , Receptores Fc , Antígenos de Histocompatibilidad Clase I

RESUMEN

Objective: This study investigates the distributional discrepancies of four single-nucleotide polymorphic loci as correlatives and causatives of dental caries susceptibility among Egyptians. Method: We conducted a cross-sectional study through the genotyping of enamelin (ENAM rs3796703), ameloblastin (AMBN rs4694075), tuftelin 1 (TUFT1 rs78802584), and kallikrein 4 (KLK4 rs2242670) in 132 adults (males = 74, females = 58) and 72 controls (males = 40, females = 32) referred from various Egyptian hospitals. For each participant, the number of decayed, missing, and filled teeth was charted, and the presence of biofilm/gingivitis/fluorosis was assessed. Bitewing radiographs were taken to detect interproximal caries. In addition, statistical analysis was conducted using Chi-square test, odds ratios, and corresponding P-values. Results: The alleles and genotypes of ENAM rs3796703, AMBN rs4694075, and KLK4 rs2242670 correlated strongly with dental caries susceptibility. However, TUFT1 rs78802584 did not exhibit such associations. Conclusion: These findings suggest the potential role of ENAM, AMBN, and KLK4 as determinants of dental caries susceptibility among Egyptian adults. The role of ENAM, AMBN, and KLK4 genetic variants is determinant in influencing susceptibility to dental caries in the Egyptian population, providing valuable insights into the genetic aspects of oral health. However, the lack of associations of caries susceptibility with TUFT1 rs78802584 contradicts its cariogenic role in many ethnicities.

RESUMEN

Porcine placental extract (PPE) is commonly used in various health foods and cosmetics. PPE use in cosmetics predominantly consist of the water-soluble fraction derived from the entire placenta. In this report, we examined the effect of the hydrophobic constituents of the PPE, specifically the sphingolipid-enriched fraction designated as the sphingolipid-enriched porcine placental extract (SLPPE), on the expression of genes associated with skin function in cultured normal human epidermal keratinocytes. Using quantitative RT-PCR (qRT-PCR) analysis, we found that SLPPE concentrations ranging from 25 to 100 µg/mL upregulated the gene expression of key components associated with the cornified envelope structure (filaggrin (FLG), involucrin (IVL) and loricrin (LOR)), cornification enzymes (transglutaminase 1 (TGM1) and TGM5) and the desquamation enzymes (kallikrein 5 (KLK5) and KLK7). Additionally, KLK5p and FLG protein (FLGp) were detected in the culture supernatants of keratinocytes treated with SLPPE at these concentrations. These findings suggest that SLPPE is possible to promote the cornification and desquamation in epidermal keratinocytes, and it may offer potential benefits in cosmetics.

Asunto(s)

Proteínas Filagrina , Calicreínas , Queratinocitos , Esfingolípidos , Transglutaminasas , Queratinocitos/efectos de los fármacos , Queratinocitos/metabolismo , Humanos , Animales , Transglutaminasas/metabolismo , Transglutaminasas/genética , Porcinos , Esfingolípidos/metabolismo , Calicreínas/metabolismo , Calicreínas/genética , Extractos Placentarios/farmacología , Células Cultivadas , Femenino , Proteínas de Filamentos Intermediarios/genética , Proteínas de Filamentos Intermediarios/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , Embarazo

RESUMEN

Human plasma kallikrein (PKa) is a member of the serine protease family and serves as a key mediator of the kallikrein-kinin system (KKS), which is known for its regulatory roles in inflammation, vasodilation, blood pressure, and coagulation. Genetic dysregulation of KKS leads to Hereditary Angioedema (HAE), which is characterized by spontaneous, painful swelling in various body regions. Importantly, HAE frequently coexists with various cancers. Despite substantial efforts towards the development of PKa inhibitors for HAE, there remains a need for bifunctional agents addressing both anti-cancer and anti-HAE aspects, especially against carcinoma-associated comorbid HAE conditions. Consequently, we investigated the therapeutic potential of the anti-glutamine prodrug, isopropyl(S)-2-((S)-2-acetamido-3-(1H-indol-3-yl)-propanamido)-6-diazo-5-oxo-hexanoate (DRP-104), and its active form, 6-Diazo-5-oxo-l-norleucine (DON), recognized for their anti-cancer properties, as novel PKa inhibitors. Utilizing structure-based in silico methods, we conducted a comparative analysis with berotralstat, a clinically approved HAE prophylactic, and sebetralstat, an investigational HAE therapeutic agent, in Phase 3 clinical trials. Inhibiting PKa with DON resulted in relatively heightened structural stability, rigidity, restricted protein folding, and solvent-accessible loop exposure, contributing to increased intra-atomic hydrogen bond formation. Conversely, PKa inhibition with DRP-104 induced restricted residue flexibility and significantly disrupted the critical SER195-HIS57 arrangement in the catalytic triad. Both DON and DRP-104, along with the reference drugs, induced strong cooperative intra-residue motion and bidirectional displacement in the PKa architecture. The results revealed favorable binding kinetics of DON/DRP-104, showing thermodynamic profiles that were either superior or comparable to those of the reference drugs. These findings support their consideration for clinical investigations into the management of carcinoma-associated HAE.

Asunto(s)

Angioedemas Hereditarios , Simulación de Dinámica Molecular , Calicreína Plasmática , Humanos , Angioedemas Hereditarios/tratamiento farmacológico , Calicreína Plasmática/antagonistas & inhibidores , Calicreína Plasmática/metabolismo , Enlace de Hidrógeno , Neoplasias/tratamiento farmacológico , Profármacos/química , Profármacos/farmacología , Profármacos/uso terapéutico , Simulación del Acoplamiento Molecular

RESUMEN

BACKGROUND: Factor (F)XII triggers contact activation by binding to foreign surfaces, with the epidermal growth factor-like 1 (EGF-1) domain being the primary binding site. Blocking FXII surface-binding might hold therapeutic value to prevent medical device-induced thrombosis. OBJECTIVES: To unravel and prevent EGF-1-mediated FXII surface-binding with a variable domain of heavy chain-only antibody (VHH). METHODS: FXII variants with glutamine substitutions of 2 positively charged amino acid patches within the EGF-1 domain were created. Their role in FXII contact activation was assessed using kaolin pull-down experiments, amidolytic activity assays, and clotting assays. FXII EGF-1 domain-specific VHHs were raised to inhibit EGF-1-mediated FXII contact activation while preserving quiescence. RESULTS: Two unique, positively charged patches in the EGF-1 domain were identified (upstream, 73K74K76K78H81K82H; downstream, 87K113K). Neutralizing the charge of both patches led to a 99% reduction in FXII kaolin binding, subsequent decrease in autoactivation of 94%, and prolongation of clot formation in activated partial thromboplastin time assays from 36 (±2) to 223 (±13) seconds. Three FXII EGF-1-specific VHHs were developed that are capable of inhibiting kaolin binding and subsequent contact system activation in plasma. The most effective VHH "F2" binds the positively charged patches and thereby dose-dependently extends activated partial thromboplastin time clotting times from 29 (±2) to 43 (±3) seconds without disrupting FXII quiescence. CONCLUSION: The 2 unique, positively charged patches in FXII EGF-1 cooperatively mediate FXII surface-binding, making both patches crucial for contact activation. Targeting these with FXII EGF-1-specific VHHs can exclusively decrease FXII surface-binding and subsequent contact activation, while preserving zymogen quiescence. These patches thus have potential as druggable targets in preventing medical device-induced thrombosis.

Asunto(s)

Coagulación Sanguínea , Factor de Crecimiento Epidérmico , Factor XII , Unión Proteica , Anticuerpos de Dominio Único , Humanos , Factor XII/metabolismo , Coagulación Sanguínea/efectos de los fármacos , Anticuerpos de Dominio Único/inmunología , Sitios de Unión , Factor de Crecimiento Epidérmico/metabolismo , Dominios Proteicos , Trombosis/prevención & control , Trombosis/inmunología , Trombosis/sangre , Relación Estructura-Actividad

RESUMEN

Prolylcarboxypeptidase (PRCP, PCP, Lysosomal Pro-X-carboxypeptidase, Angiotensinase C) controls angiotensin- and kinin-induced cell signaling. Elevation of PRCP appears to be activated in chronic inflammatory diseases [cardiovascular disease (CVD), diabetes] in proportion to severity. Vascular endothelial cell senescence and mitochondrial dysfunction have consistently been shown in models of CVD in aging. Cellular senescence, a driver of age-related dysfunction, can differentially alter the expression of lysosomal enzymes due to lysosomal membrane permeability. There is a lack of data demonstrating the effect of age-related dysfunction on the expression and function of PRCP. To explore the changes in PRCP, the PRCP-dependent prekallikrein (PK) pathway was characterized in early- and late-passage human pulmonary artery endothelial cells (HPAECs). Detailed kinetic analysis of cells treated with high molecular weight kininogen (HK), a precursor of bradykinin (BK), and PK revealed a mechanism by which senescent HPAECs activate the generation of kallikrein upon the assembly of the HK-PK complex on HPAECs in parallel with an upregulation of PRCP and endothelial nitric oxide (NO) synthase (eNOS) and NO formation. The NO production and expression of both PRCP and eNOS increased in early-passage HPAECs and decreased in late-passage HPAECs. Low activity of PRCP in late-passage HPAECs was associated with rapid decreased telomerase reverse transcriptase mRNA levels. We also found that, with an increase in the passage number of HPAECs, reduced PRCP altered the respiration rate. These results indicated that aging dysregulates PRCP protein expression, and further studies will shed light into the complexity of the PRCP-dependent signaling pathway in aging.

Asunto(s)

Biomarcadores , Carboxipeptidasas , Senescencia Celular , Células Endoteliales , Humanos , Células Endoteliales/metabolismo , Biomarcadores/metabolismo , Carboxipeptidasas/metabolismo , Carboxipeptidasas/genética , Precalicreína/metabolismo , Precalicreína/genética , Bradiquinina/farmacología , Bradiquinina/metabolismo , Arteria Pulmonar/metabolismo , Arteria Pulmonar/citología , Células Cultivadas , Quininógeno de Alto Peso Molecular/metabolismo , Transducción de Señal , Óxido Nítrico Sintasa de Tipo III/metabolismo , Óxido Nítrico Sintasa de Tipo III/genética , Calicreínas/metabolismo , Calicreínas/genética

RESUMEN

Hereditary angioedema (HAE) due to C1-inhibitor deficiency is a rare, debilitating, genetic disorder characterized by recurrent, unpredictable, attacks of edema. The clinical symptoms of HAE arise from excess bradykinin generation due to dysregulation of the plasma kallikrein-kinin system (KKS). A quantitative systems pharmacology (QSP) model that mechanistically describes the KKS and its role in HAE pathophysiology was developed based on HAE attacks being triggered by autoactivation of factor XII (FXII) to activated FXII (FXIIa), resulting in kallikrein production from prekallikrein. A base pharmacodynamic model was constructed and parameterized from literature data and ex vivo assays measuring inhibition of kallikrein activity in plasma of HAE patients or healthy volunteers who received lanadelumab. HAE attacks were simulated using a virtual patient population, with attacks recorded when systemic bradykinin levels exceeded 20 pM. The model was validated by comparing the simulations to observations from lanadelumab and plasma-derived C1-inhibitor clinical trials. The model was then applied to analyze the impact of nonadherence to a daily oral preventive therapy; simulations showed a correlation between the number of missed doses per month and reduced drug effectiveness. The impact of reducing lanadelumab dosing frequency from 300 mg every 2 weeks (Q2W) to every 4 weeks (Q4W) was also examined and showed that while attack rates with Q4W dosing were substantially reduced, the extent of reduction was greater with Q2W dosing. Overall, the QSP model showed good agreement with clinical data and could be used for hypothesis testing and outcome predictions.

RESUMEN

Intervertebral disc degeneration (IVDD) is a leading cause of degenerative spinal disorders, involving complex biological processes. This study investigates the role of the kallikrein-kinin system (KKS) in IVDD, focusing on the protective effects of bradykinin (BK) on nucleus pulposus cells (NPCs) under oxidative stress. Clinical specimens were collected, and experiments were conducted using human and rat primary NPCs to elucidate BK's impact on tert-butyl hydroperoxide (TBHP)-induced oxidative stress and damage. The results demonstrate that BK significantly inhibits TBHP-induced NPC apoptosis and restores mitochondrial function. Further analysis reveals that this protective effect is mediated through the BK receptor 2 (B2R) and its downstream PI3K/AKT pathway. Additionally, BK/PLGA sustained-release microspheres were developed and validated in a rat model, highlighting their potential therapeutic efficacy for IVDD. Overall, this study sheds light on the crucial role of the KKS in IVDD pathogenesis and suggests targeting the B2R as a promising therapeutic strategy to delay IVDD progression and promote disc regeneration.

Asunto(s)

Apoptosis , Bradiquinina , Degeneración del Disco Intervertebral , Núcleo Pulposo , Estrés Oxidativo , Ratas Sprague-Dawley , terc-Butilhidroperóxido , Animales , Núcleo Pulposo/efectos de los fármacos , Núcleo Pulposo/patología , Núcleo Pulposo/metabolismo , terc-Butilhidroperóxido/toxicidad , Degeneración del Disco Intervertebral/tratamiento farmacológico , Degeneración del Disco Intervertebral/patología , Humanos , Masculino , Bradiquinina/farmacología , Apoptosis/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Ratas , Células Cultivadas , Receptor de Bradiquinina B2/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Femenino , Microesferas , Transducción de Señal/efectos de los fármacos , Copolímero de Ácido Poliláctico-Ácido Poliglicólico/química , Fosfatidilinositol 3-Quinasas/metabolismo , Modelos Animales de Enfermedad

RESUMEN

Glycolic acid (GA) is extensively used in cosmetic formulations and skin peeling treatments but its adverse effects, notably severe disruption of epidermal structure, limit its clinical utility. However, the detailed impact of GA on epidermal homeostasis, including changes in structure and protein expression over time, is not fully understood. This study employed a reconstructed human epidermis (RHE) model to assess the effects of varying GA concentrations on epidermal proliferation, differentiation, and desquamation at different time points. Through histology, immunofluorescence, and immunohistochemistry, we observed that 35% GA concentration adversely caused abnormal epidermal homeostasis by affecting epidermal proliferation, differentiation and desquamation. Our findings reveal time-specific responses of key proteins to GA: Filaggrin, Involucrin, Loricrin, and Ki67 showed very early responses; KLK10 an early response; and AQP3 and K10 late responses. This research provides a detailed characterization of GA's effects in an RHE model, mimicking clinical superficial peeling and identifying optimal times for detecting GA-induced changes. Our results offer insights for designing interventions to mitigate GA's adverse effects on skin, enhancing the safety and efficacy of GA peeling treatments.

Asunto(s)

Proliferación Celular , Epidermis , Proteínas Filagrina , Glicolatos , Homeostasis , Glicolatos/toxicidad , Humanos , Epidermis/efectos de los fármacos , Epidermis/metabolismo , Homeostasis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Factores de Tiempo , Queratinocitos/efectos de los fármacos , Queratinocitos/metabolismo

RESUMEN

BACKGROUND: Hereditary angioedema (HAE) is a genetic disorder that manifests as recurrent angioedema attacks, most frequently due to absent or reduced C1 inhibitor (C1INH) activity. C1INH is a crucial regulator of enzymatic cascades in the complement, fibrinolytic, and contact systems. Inter-α-trypsin inhibitor heavy chain 4 (ITIH4) is an abundant plasma protease inhibitor that can inhibit enzymes in the proteolytic pathways associated with HAE. Nothing is known about its role in HAE. OBJECTIVE: We investigated ITIH4 activation in HAE, establishing it as a potential biomarker, and explored its involvement in HAE-associated proteolytic pathways. METHODS: Specific immunoassays for noncleaved ITIH4 (intact ITIH4) and an assay detecting both intact and cleaved ITIH4 (total ITIH4) were developed. We initially tested serum samples from HAE patients (n = 20), angiotensin-converting enzyme inhibitor-induced edema patients (ACEI) (n = 20), and patients with HAE of unknown cause (HAE-UNK) (n = 20). Validation involved an extended cohort of 80 HAE patients (60 with HAE-C1INH type 1, 20 with HAE-C1INH type 2), including samples taken during attack and quiescent disease periods, as well as samples from 100 healthy controls. RESULTS: In 63% of HAE patients, intact ITIH4 assay showed lower signals than total ITIH4 assay. This difference was not observed in ACEI and HAE-UNK patients. Western blot analysis confirmed cleaved ITIH4 with low intact ITIH4 samples. In serum samples lacking intact endogenous ITIH4, we observed immediate cleavage of added recombinant ITIH4, suggesting continuous enzymatic activity in the serum. Confirmatory HAE cohort analysis revealed significantly lower intact ITIH4 levels in both type 1 and type 2 HAE patients compared to controls, with consistently low intact/total ITIH4 ratios during clinical HAE attacks. CONCLUSION: The disease-specific low intact ITIH4 levels highlight its unique nature in HAE. ITIH4 may exhibit compensatory mechanisms in HAE, suggesting its utility as a diagnostic and prognostic biomarker. The variations during quiescent and active disease periods raise intriguing questions about the dynamics of proteolytic pathways in HAE.

Asunto(s)

Angioedemas Hereditarios , Biomarcadores , Proteínas Inhibidoras de Proteinasas Secretoras , Humanos , Angioedemas Hereditarios/diagnóstico , Angioedemas Hereditarios/tratamiento farmacológico , Angioedemas Hereditarios/sangre , Femenino , Masculino , Adulto , Persona de Mediana Edad , Biomarcadores/sangre , Anciano , Adolescente , Adulto Joven , Glicoproteínas/sangre , Proteína Inhibidora del Complemento C1/genética