Protease-activated Receptor Research Articles

The Role of Tissue Factor In Signaling Pathways of Pathological Conditions and Angiogenesis.

Tissue factor (TF) is an integral transmembrane protein associated with the extrinsic coagulation pathway. TF gene expression is regulated in response to inflammatory cytokines, bacterial lipopolysaccharides, and mechanical injuries. TF activity may be affected by phosphorylation of its cytoplasmic domain and alternative splicing. TF acts as the primary initiator of physiological hemostasis, which prevents local bleeding at the injury site. However, aberrant expression of TF, accompanied by the severity of diseases and infections under various pathological conditions, triggers multiple signaling pathways that support thrombosis, angiogenesis, inflammation, and metastasis. Protease-activated receptors (PARs) are central in the downstream signaling pathways of TF. In this study, we have reviewed the TF signaling pathways in different pathological conditions, such as wound injury, asthma, cardiovascular diseases (CVDs), viral infections, cancer and pathological angiogenesis. Angiogenic activities of TF are critical in the repair of wound injuries and aggressive behavior of tumors, which are mainly performed by the actions of vascular endothelial growth factor (VEGF) and hypoxia-inducible factor-1 (HIF1-α). Pro-inflammatory effects of TF have been reported in asthma, CVDs and viral infections, including COVID-19, which result in tissue hypertrophy, inflammation, and thrombosis. TF-FVII induces angiogenesis via clotting-dependent and -independent mechanisms. Clottingdependent angiogenesis is induced via the generation of thrombin and cross-linked fibrin network, which facilitate vessel infiltration and also act as a reservoir for endothelial cells (ECs) growth factors. Expression of TF in tumor cells and ECs triggers clotting-independent angiogenesis through induction of VEGF, urokinase-type plasminogen activator (uPAR), early growth response 1 (EGR1), IL8, and cysteine-rich angiogenic inducer 61 (Cyr61).

Abstract 43: PAR1 Inhibition with Vorapaxar Enhances Renal Function and Modulates Inflammation Mediators in Diabetic Nephropathy

Diabetic nephropathy (DN) is the leading cause of end-stage renal failure, and hypertension is a comorbidity in about 90% of all cases. Characteristics of pathological changes of DN include severe albuminuria, renal hyperfiltration, glomerular basement membrane thickening, and glomerulosclerosis. Identifying the molecular mechanisms underlying DN is critical for developing more effective therapeutic approaches. Thus, interventions targeting podocytes have become a major focus for diseases like DN due to their crucial role in regulating glomerular permeability and maintaining glomerular structures. Our previous studies have demonstrated that serine proteases and Protease-activated receptor 1 (PAR1) are involved in developing DN through TRPC6-mediated calcium signaling in podocytes. Utilizing a PAR1 heterozygous knockout rat model with a type 2 diabetic background, we showed that reducing PAR1 expression decreases microalbuminuria and glomerular damage. This study aims to define the effects and potential mechanisms of pharmacological blockade of PAR1 on the development of DN. To achieve this, 16-week-old T2DN rats were treated with a potent and selective PAR1 antagonist (vorapaxar (SCH 530348), 10 mg/kg of diet) for 16 weeks to determine the effect of PAR1 inhibition. We found improvements in glomerular filtration rate (GFR) in treated animals compared to control after 16 weeks of treatment (0.8 ± 0.2 vs 1.2 ± 0.2 mL/min/100g b.w., p=0.029, N≥4). We observed a trend toward differences in basal intracellular calcium concentration in podocytes between the control and treated groups, although these changes were not statistically significant (202 ± 95 vs 152 ± 75 nM, p=0.19, n≥10, N=3). Additionally, inhibition of PAR1 caused modulation in the inflammation-resolution mediators in the kidney with significant upregulation of Maresins Conjugates in Tissue Regeneration (MCTR1) and downregulation of hydroxy-docosahexaenoic acids (7-,14-,17-HDHA). Changes in GFR may be linked to reduced glomerular damage, indicating that the structural integrity of the glomeruli is better maintained in treated animals. Alterations in the lipidomic profile could indicate a reduction in the inflammatory processes typically associated with DN. Overall, our data indicates that vorapaxar improves glomerular filtration rate and modulates lipid mediators in a type 2 diabetic rat model. This suggests that targeting PAR1 could be a promising therapeutic strategy for diabetic nephropathy.

Liposomal fusion of plant-based extracellular vesicles to enhance skin anti-inflammation

This study reports a hybrid extracellular vesicle (EV)/liposome system developed through vesicular membrane fusion between anti-inflammatory Hydrangea macrophylla leaf EVs (HML-EVs) and terpinen-4-ol/azelamide monoethanolamine-loaded liposomes. Successful liposomal fusion between HML-EVs and terpinen-4-ol/azelamide monoethanolamine-loaded-liposome membranes, confirmed using the two-color fluorescence resonance energy transfer mechanism, enabled the scaled production of a stable hybrid HML-EV/liposome dispersion at a length scale of 100–200 nm. We show that the hybrid EV/liposome significantly inhibits apoptosis by nitric oxide and tumor necrosis factor-α production. Furthermore, HML-EV/liposomes can inhibit the synthesis of protease-activated receptor-2, interleukin-6, c-Jun, and thymic stromal lymphopoietin, thus exhibiting anti-inflammatory performance. These findings highlight that the hybrid EV/liposome system established in this study alleviates skin irritation and improves skin anti-inflammation.

Protease activated receptor-1 regulates mixed lineage kinase-3 to drive triple-negative breast cancer tumorigenesis

Triple-negative breast cancer (TNBC) is difficult to treat breast cancer subtype due to lack or insignificant expressions of targetable estrogen receptor (ER) and human epidermal growth factor receptor 2 (HER2). Therefore, finding a targetable protein or signaling pathway in TNBC would impact patient care. Here, we report that a member of the Mixed Lineage Kinase (MLK) family, MLK3, is an effector of G-protein-coupled protease-activated receptors 1 (PAR1) and targeting MLK3 by a small-molecule inhibitor prevented PAR1-mediated TNBC tumorigenesis. In silico and immunohistochemistry analysis of human breast tumors showed overexpression of PAR1 and MLK3 in TNBC tumors. Treating α-thrombin and PAR1 agonist increased MLK3 and JNK activities and induced cell migration in TNBC cells. The PAR1 positive/high (PAR1+/hi) population of TNBC cells showed aggressive tumor phenotype with increased MLK3 signaling. Moreover, combined inhibition of the PAR1 and MLK3 mitigated the TNBC tumor burden in preclinical TNBC models. Our data suggests that activation of the PAR1-MLK3 axis promotes TNBC tumorigenesis. Therefore, combinatorial therapy targeting MLK3 and PAR1 could effectively reduce TNBC tumor burden.

Long-term GABA Supplementation Regulates Diabetic Gastroenteropathy through GABA Receptor/trypsin-1/PARs/Akt/COX-2 Axis.

Molecular alterations of diabetic gastroenteropathy are poorly identified. This study investigates the effects of prolonged GABA supplementation on key protein expression levels of trypsin-1, PAR-1, PAR-2, PAR-3, PI3K, Akt, COX-2, GABAA, and GABAB receptors in the gastric tissue of type 2 diabetic rats (T2DM). To induce T2DM, a 3-month high-fat diet and 35 mg/kg of streptozotocin was used. Twenty-four male Wistar rats were divided into 4 groups: (1) control, (2) T2DM, (3) insulin-treated (2.5 U/kg), and (4)GABA-treated (1.5 g/kg GABA). Blood glucose was measured weekly. The protein expressions were assessed using western blotting. Histopathological changes were examined by H&E and Masson's staining. Diabetic rats show reduced NOS1 and elevated COX-2 and trypsin-1 protein expression levels in gastric tissue. Insulin and GABA therapy restored the NOS1 and COX-2 levels to control values. Insulin treatment increased PI3K, Akt, and p-Akt and, decreased trypsin-1, PAR-1, PAR-2, and PAR-3 levels in the diabetic rats. Levels of GABAA and GABAB receptors normalized following insulin and GABA therapy. H&E staining indicated an increase in mucin secretion following GABA treatment. These results suggest that GABA by acting on GABA receptors may regulate the trypsin-1/PARs/Akt/COX-2 pathway and thereby improve complications of diabetic gastroenteropathy.

DIFFERENCES IN PROTEASE ACTIVATED RECEPTOR-1 AND THROMBINE LEVELS IN PREECLAMPSIA AND NORMAL PREGNANCY

Introduction: Preeclampsia is a condition caused by alterations in endothelial function during pregnancy. Changes in endothelial function result in an increase in coagulation and microvascular fibrin accumulation, which results in impaired placental perfusion. Thrombin, which converts fibrin to fibrinogen, as well as platelet activity, the fibrinolytic system, and anticoagulants, are all procoagulant circumstances in preeclampsia. Thrombin contributes to the pathogenesis of preeclampsia by increasing the expression of sFlt-1 thereby providing an antiangiogenic response. Protease Activated Receptor-1 (PAR-1) is a mediator of thrombin for coagulation and inflammation in preeclampsia. Inhibition of Protease Activated Receptor-1 expression in trophoblasts can enhance placental angiogenesis and vascular remodeling. Recently, only few studies have assessed the levels of Protease Activated Receptor -7 and thrombin in preeclampsia.Objective: To determine the difference in levels of Protease Activated Receptor-1 and thrombin in preeclampsia and normal pregnancyMethods: This study is observational with a cross-sectional comparative study design. Sampling was conducted from March 2020 to March 2021. A total of 66 patients were investigated, with 33 samples of preeclampsia and 33 samples of normal pregnancy. The independent sample T-test was used for statistical analysis.Results: The mean levels of Protease Activated Receptor-1 in the preeclampsia group were higher at 28.56 ± 7.68 ng/mL while normal pregnancy was 21.67 ± 6.92 ng/mL. The results of statistical tests showed that there was a significant difference in levels of Protease Activated Receptor-1 between the preeclampsia and normal pregnancy groups (p<0.05). The mean thrombin level in the preeclampsia group was higher at 72.23 ± 7.99 ng/mL, while in normal pregnancy it was 63.70 ± 8.92 ng/mL. The difference in thrombin levels between the preeclampsia and normal pregnancy groups was statistically significant (p<0.05).Conclusion: Preeclampsia was associated with greater levels of Protease Activated Receptor-1 and thrombin than normal pregnancy. There was a significant difference in the mean levels of Protease Activated Receptor-1 and thrombin between preeclampsia and normal pregnancy.Keywords: Thrombin, Protease Activated Receptor-1(PAR-1), Preeclampsia

The biochemistry of melanogenesis: an insight into the function and mechanism of melanogenesis-related proteins.

Melanin is an amino acid derivative produced by melanocyte through a series of enzymatic reactions using tyrosinase as substrate. Human skin and hair color is also closely related to melanin, so understanding the mechanisms and proteins that produce melanin is very important. There are many proteins involved in the process of melanin expression, For example, proteins involved in melanin formation such as p53, HNF-1α (Hepatocyte nuclear factor 1α), SOX10 (Sry-related HMg-Box gene 10) and pax3 (paired box gene 3), MC1R(Melanocortin 1 Receptor), MITF (Microphthalmia-associated transcription factor), TYR (tyrosinase), TYRP1 (tyrosinase-related protein-1), TYRP2 (tyrosinase-related protein-2), and can be regulated by changing their content to control the production rate of melanin. Others, such as OA1 (ocular albinism type 1), Par-2 (protease-activated receptor 2) and Mlph (Melanophilin), have been found to control the transfer rate of melanosomes from melanocytes to keratinocytes, and regulate the amount of human epidermal melanin to control the depth of human skin color. In addition to the above proteins, there are other protein families also involved in the process of melanin expression, such as BLOC, Rab and Rho. This article reviews the origin of melanocytes, the related proteins affecting melanin and the basic causes of related gene mutations. In addition, we also summarized the active ingredients of 5 popular whitening cosmetics and their mechanisms of action.

Mechanisms and Therapeutic Targets of Staphylococcus Aureus - Induced Itch in Atopic Dermatitis

Atopic dermatitis is a chronic skin disorder marked by inflammation, erythema, and pruritus, often beginning in childhood and characterized by exacerbations and remissions. Its etiology involves genetic, immunological, and environmental factors. Staphylococcus aureus, a common pathogen, exacerbates atopic dermatitis by producing toxins and enzymes such as the serine protease V8, which activates the protease-activated receptor 1 (PAR1) on sensory neurons, inducing itch. This mechanism highlights potential therapeutic targets for alleviating pruritus. Research into the physiological pathways of itch, including the role of PAR1 and other protease-activated receptors, reveals promising strategies for treatment. PAR1 antagonists could be repurposed to treat chronic itch, providing new therapeutic avenues. PAR1 and other protease-activated receptors, is crucial in developing new treatment strategies. Anti-pruritic therapies targeting these pathways, such as PAR1 antagonists, show promise in mitigating itch symptoms. Moreover, existing drugs that inhibit PAR1 could be repurposed for treating chronic itch, providing a new avenue for relief in patients with atopic dermatitis. Additionally, the evolutionary role of itch induced by microorganisms suggests pathogens may exploit neural reflexes to enhance their spread. Advancements in understanding the mechanisms behind S. aureus-induced itch and the physiological pathways involved offer promising new directions for therapeutic intervention. Advancements in understanding the mechanisms of Staphylococcal aureus-induced itch and associated physiological pathways offer promising directions for therapeutic intervention, potentially improving management and treatment outcomes for patients with atopic dermatitis and other pruritic conditions.

Beyond Anticoagulation: A Comprehensive Review of Non-Vitamin K Oral Anticoagulants (NOACs) in Inflammation and Protease-Activated Receptor Signaling.

Non-vitamin K oral anticoagulants (NOACs) have revolutionized anticoagulant therapy, offering improved safety and efficacy over traditional agents like warfarin. This review comprehensively examines the dual roles of NOACs-apixaban, rivaroxaban, edoxaban, and dabigatran-not only as anticoagulants, but also as modulators of inflammation via protease-activated receptor (PAR) signaling. We highlight the unique pharmacotherapeutic properties of each NOAC, supported by key clinical trials demonstrating their effectiveness in preventing thromboembolic events. Beyond their established anticoagulant roles, emerging research suggests that NOACs influence inflammation through PAR signaling pathways, implicating factors such as factor Xa (FXa) and thrombin in the modulation of inflammatory responses. This review synthesizes current evidence on the anti-inflammatory potential of NOACs, exploring their impact on inflammatory markers and conditions like atherosclerosis and diabetes. By delineating the mechanisms by which NOACs mediate anti-inflammatory effects, this work aims to expand their therapeutic utility, offering new perspectives for managing inflammatory diseases. Our findings underscore the broader clinical implications of NOACs, advocating for their consideration in therapeutic strategies aimed at addressing inflammation-related pathologies. This comprehensive synthesis not only enhances understanding of NOACs' multifaceted roles, but also paves the way for future research and clinical applications in inflammation and cardiovascular health.

Attenuation of Atherosclerosis with PAR4 Deficiency: Differential Platelet Outcomes in apoE -/- vs. Ldlr -/- Mice.

Cardiovascular disease (CVD) is a significant burden globally and, despite current therapeutics, remains the leading cause of death. Platelet inhibitors are of interest in CVD treatment to reduce thrombus formation post-plaque rupture as well their contribution to inflammation throughout the progression of atherosclerosis. Protease activated receptor 4 (PAR4) is a receptor highly expressed by platelets, strongly activated by thrombin, and plays a vital role in platelet activation and aggregation. However, the role of PAR4. Mice on a low-density lipoprotein receptor-deficient ( Ldlr -/- ) background were bred with Par4 deficient ( Par4 -/- ) mice to create Ldlr -/- /Par4 +/+ and Ldlr -/- /Par4 -/- cousin lines. Mice were fed high fat (42%) and cholesterol (0.2%) 'Western' diet for 12 weeks for all studies. Bone marrow transplant (BMT) studies were conducted by irradiating Ldlr -/- /Par4 +/+ and Ldlr -/- /Par4 -/- mice with 550 rads (2x, 4 hours apart) and then repopulated with Par4 +/+ or Par4 -/- bone marrow. To determine if the effects of thrombin were mediated solely by PAR4, the thrombin inhibitor dabigatran was added to the 'Western' diet. Ldlr -/- /Par4 -/- given dabigatran did not further decrease their atherosclerotic burden. Differences between apolipoprotein E deficient ( apoE -/- ) and Ldlr -/- platelets were assessed for changes in reactivity. We observed higher PAR4 abundance in arteries with atherosclerosis in human and mice versus healthy controls. PAR4 deficiency attenuated atherosclerosis in the aortic sinus and root versus proficient controls. BMT studies demonstrated this effect was due to hematopoietic cells, most likely platelets. PAR4 appeared to be acting independent of PAR1, as there werer no changes with addition of dabigatran to PAR4 deficient mice. apoE -/- platelets are hyperreactive compared to Ldlr -/- platelets. Hematopoietic-derived PAR4, most likely platelets, plays a vital role in the development and progression of atherosclerosis. Specific targeting of PAR4 may be a potential therapeutic target for CVD. Deficiency of protease-activated receptor 4 attenuates the development of diet-induced atherosclerosis in a Ldlr -/- mouse model. PAR4 deficiency in hematopoietic cells is atheroprotective. PAR4 deficiency accounts for the majority of thrombin-induced atherosclerosis in a Ldlr -/- mouse model. The examination of platelet-specific proteins and platelet activation should be carefully considered before using the apoE -/- or Ldlr -/- mouse models of atherosclerosis.

Elucidating PAR1 as a therapeutic target for delayed traumatic brain injury: Unveiling the PPAR-γ/Nrf2/HO-1/GPX4 axis to suppress ferroptosis and alleviate NLRP3 inflammasome activation in rats

Repetitive traumatic brain injury (RTBI) is acknowledged as a silent overlooked public health crisis, with an incomplete understanding of its pathomechanistic signaling pathways. Mounting evidence suggests the involvement of thrombin and its receptor, the protease-activated receptor (PAR)1, in the development of secondary injury in TBI; however, the consequences of PAR1 modulation and its impact on ferroptosis-redox signaling, and NLRP3 inflammasome activation in RTBI, remain unclear. Further, the utilitarian function of PAR1 as a therapeutic target in RTBI has not been elucidated. To study this crosstalk, RTBI was induced in Wistar rats by daily weight drops on the right frontal region for five days. Three groups were included: normal control, untreated RTBI, and RTBI+SCH79797 (a PAR1 inhibitor administered post-trauma at 25 μg/kg/day). The concomitant treatment of PAR1 antagonism improved altered behavior function, cortical histoarchitecture, and neuronal cell survival. Moreover, the receptor blockade downregulated mRNA expression of PAR1 but upregulatedthat of the neuroprotective receptor PPAR-γ. The anti-inflammatory impact of SCH79797 was signified by the low immune expression/levels of NF-κB p65,TNF-α, IL-1β, and IL-18. Consequently, the PAR1 blocker hindered the formation of inflammasome components NLRP3, ASC, and activated caspase-1. Ultimately, SCH79797 treatment abated ferroptosis-dependent iron redox signaling through the activation of the antioxidant Nrf2/HO-1 axis and its subsequent antioxidant machinery (GPX4, SOD) to limit lipid peroxidation, iron accumulation, and transferrin serum increment. Collectively, SCH79797 offered putative preventive mechanisms against secondary RTBI consequences in rats by impeding ferroptosis and NLRP3 inflammasome through activating the PPAR-γ/Nrf2 antioxidant cue.

FXa-mediated PAR-2 promotes the efficacy of immunotherapy for hepatocellular carcinoma through immune escape and anoikis resistance by inducing PD-L1 transcription

BackgroundThe high metastasis rate is one of the main reasons for the poor prognosis of patients with hepatocellular carcinoma (HCC). Coagulation factor Xa (FXa) and its receptor proteinase-activated receptor-2 (PAR-2)...

New Target(s) for RNF43 Regulation: Implications for Therapeutic Strategies.

Cancer cells depend on specific oncogenic pathways or present a genetic alteration that leads to a particular disturbance. Still, personalized and targeted biological therapy remains challenging, with current efforts generally yielding disappointing results. Carefully assessing onco-target molecular pathways can, however, potently assist with such efforts for the selection of patient populations that would best respond to a given drug treatment. RNF43, an E3 ubiquitin ligase that negatively regulates Wnt/frizzled (FZD) receptors by their ubiquitination, internalization, and degradation, controls a key pathway in cancer. Recently, additional target proteins of RNF43 were described, including p85 of the PI3K/AKT/mTOR signaling pathway and protease-activated receptor 2 (PAR2), a G-protein-coupled receptor that potently induces β-catenin stabilization, independent of Wnts. RNF43 mutations with impaired E3 ligase activity were found in several types of cancers (e.g., gastrointestinal system tumors and endometrial and ovarian cancer), pointing to a high dependency on FZD receptors and possibly PAR2 and the PI3K/AKT/mTOR signaling pathway. The development of drugs toward these targets is essential for improved treatment of cancer patients.

Inhibition of Protease-Activated Receptor-2 Activation in Parkinson's Disease Using 1-Piperidin Propionic Acid.

In Parkinson's disease, neuroinflammation is a double-edged sword; when inflammation occurs it can have harmful effects, despite its important role in battling infections and healing tissue. Once triggered by microglia, astrocytes acquire a reactive state and shift from supporting the survival of neurons to causing their destruction. Activated microglia and Proteinase-activated receptor-2 (PAR2) are key points in the regulation of neuroinflammation. 1-Piperidin Propionic Acid (1-PPA) has been recently described as a novel inhibitor of PAR2. The aim of our study was to evaluate the effect of 1-PPA in neuroinflammation and microglial activation in Parkinson's disease. Protein aggregates and PAR2 expression were analyzed using Thioflavin S assay and immunofluorescence in cultured human fibroblasts from Parkinson's patients, treated or untreated with 1-PPA. A significant decrease in amyloid aggregates was observed after 1-PPA treatment in all patients. A parallel decrease in PAR2 expression, which was higher in sporadic Parkinson's patients, was also observed both at the transcriptional and protein level. In addition, in mouse LPS-activated microglia, the inflammatory profile was significantly downregulated after 1-PPA treatment, with a remarkable decrease in IL-1β, IL-6, and TNF-α, together with a decreased expression of PAR2. In conclusion, 1-PPA determines the reduction in neuroglia inflammation and amyloid aggregates formation, suggesting that the pharmacological inhibition of PAR2 could be proposed as a novel strategy to control neuroinflammation.

Structural basis of tethered agonism and G protein coupling of protease-activated receptors

Protease-activated receptors (PARs) are a unique group within the G protein-coupled receptor superfamily, orchestrating cellular responses to extracellular proteases via enzymatic cleavage, which triggers intracellular signaling pathways. Protease-activated receptor 1 (PAR1) is a key member of this family and is recognized as a critical pharmacological target for managing thrombotic disorders. In this study, we present cryo-electron microscopy structures of PAR1 in its activated state, induced by its natural tethered agonist (TA), in complex with two distinct downstream proteins, the Gq and Gi heterotrimers, respectively. The TA peptide is positioned within a surface pocket, prompting PAR1 activation through notable conformational shifts. Contrary to the typical receptor activation that involves the outward movement of transmembrane helix 6 (TM6), PAR1 activation is characterized by the simultaneous downward shift of TM6 and TM7, coupled with the rotation of a group of aromatic residues. This results in the displacement of an intracellular anion, creating space for downstream G protein binding. Our findings delineate the TA recognition pattern and highlight a distinct role of the second extracellular loop in forming β-sheets with TA within the PAR family, a feature not observed in other TA-activated receptors. Moreover, the nuanced differences in the interactions between intracellular loops 2/3 and the Gα subunit of different G proteins are crucial for determining the specificity of G protein coupling. These insights contribute to our understanding of the ligand binding and activation mechanisms of PARs, illuminating the basis for PAR1’s versatility in G protein coupling.

Reduced platelet activation and thrombus formation in male transgenic model mice of Alzheimer's disease suggests early sex-specific differences in platelet pathophysiology

Alzheimer's disease (AD) is the most common form of dementia and characterized by extracellular amyloid-β (Aβ) plaques, intracellular neurofibrillary tau tangles and neurodegeneration. Over 80 % of AD patients also exhibit cerebral amyloid angiopathy (CAA). CAA is a cerebrovascular disease caused by deposition of Aβ in the walls of cerebral blood vessels leading to vessel damage and impairment of normal blood flow. To date, different studies suggest that platelet function, including activation, adhesion and aggregation, is altered in AD due to vascular Aβ deposition. For example, the transgenic AD model mice APP23 mice that exhibit CAA and parenchymal Aβ plaques, show pre-activated platelets in the blood circulation and increased platelet integrin activation leading to a pro-thrombotic phenotype in these mice late stages of AD. However, it is still an open question whether or not platelets exhibit changes in their activation profile before they are exposed to vascular Aβ deposits. Therefore, the present study examined platelets from middle-aged transgenic APP23 mice at the age of 8–10 months. At this age, APP23 mice show amyloid plaques in the brain parenchyma but not in the vasculature. Our analyses show that these APP23 mice have unaltered platelet numbers and size, and unaltered surface expression of glycoproteins. However, the number of dense granules in transgenic platelets was increased while the release was unaltered. Male, but not female APP23 mice, exhibited reduced platelet activation after stimulation of the thrombin receptor PAR4 and decreased thrombus stability on collagen under flow conditions ex vivo compared to control mice. In an arterial thrombosis model in vivo, male APP23 mice showed attenuated occlusion of the injured artery compared to controls. These findings provide clear evidence for early changes in platelet activation and thrombus formation in male mice before development of overt CAA. Furthermore, reduced platelet activation and thrombus formation suggest sex-specific differences in platelet physiology in AD that has to be considered in future studies of platelets and their role in AD.

Postpartum acute myometritis suppresses expression of contraction-associated proteins in the gravid uterus

Uterine atony is a major contributor to postpartum hemorrhage. We previously proposed the novel histological concept of postpartum acute myometritis (PAM) to elucidate the pathophysiology of uterine atony. This concept involves the infiltration of macrophages and neutrophils, as well as mast cell and complement activation in the myometrium. However, the pathological mechanism underlying uterine atony in the context of PAM remains unclear. Herein, we focused on uterine contraction-associated proteins (CAPs) including connexin 43 (Cx43), oxytocin receptors (OXR), prostaglandin receptors EP1, EP3, FP, and protease-activated receptor (PAR)-1. This follow-up study aimed to compare CAP expression between PAM and control groups. We selected 38 PAM subjects from the cases enrolled in our amniotic fluid embolism registry between 2011 and 2018. Control tissues from 10 parturients were collected during cesarean section. We stained the myometrial tissues with the following CAP markers, inflammatory cell markers, and other markers: Cx43, OXR, EP1, EP3, FP, PAR-1, C5a receptor, tryptase, neutrophil elastase, CD68, β-actin, and Na+/K+-ATPase. The immunostaining-positive areas of Cx43, OXR, EP1, EP3, and FP standardized by β-actin in the PAM tissue were significantly smaller than in the control group, whereas those of PAR-1 and Na+/K+-ATPase increased significantly in the PAM group. The Cx43- and OXR-positive areas correlated negatively with the immunostaining-positive cell numbers of CD68 and tryptase with halo, respectively. PAM may impair individual and synchronized myocyte contraction, leading to uterine atony refractory to uterotonics. Further cell-based studies are needed to elucidate the molecular mechanism by which inflammatory cells suppress CAP expression.

The involvement of keratinocytes in pruritus of chronic inflammatory dermatosis.

Frequent itching and incessant scratching are commonly observed in various chronic inflammatory skin conditions, including atopic dermatitis and psoriasis. The persistent and prolonged nature of pruritus can worsen one's quality of life. Keratinocytes (KCs), the predominant cells of the epidermis, have been confirmed to interact with sensory neurons and immune cells and be involved in chronic skin inflammatory diseases associated with pruritus. Initially, KCs and sensory neurons form a unique synapse-like connection within the epidermis, serving as the structural foundation for their interaction. Additionally, several receptors, including toll-like receptors and protease-activated receptor 2, expressed on KCs, become activated in an inflammatory milieu. On the one hand, activated KCs are sources of pro-inflammatory cytokines and neurotrophic factors, such as adenosine triphosphate, thymic stromal lymphopoietin, and nerve growth factor, which directly or indirectly participate in stimulating sensory neurons, thereby contributing to the itch sensations. On the other hand, KCs also function as primary transducers alongside intraepidermal nerve endings, directly initiating pruritic responses. This review summarizes the current literature and highlights the critical role of KCs in the development and persistence of chronic itch in inflammatory skin disorders.

Molecular Mechanisms Underlying Loss of Vascular and Epithelial Integrity in Irritable Bowel Syndrome

The pathophysiology of irritable bowel syndrome (IBS) is multifactorial and includes epithelial barrier dysfunction, a key element at the interface between the gut lumen and the deeper intestinal layers. Beneath the epithelial barrier there is the vascular one representing the last barrier to avoid luminal antigen dissemination The aims of this study were to correlate morpho-functional aspects of epithelial and vascular barriers with symptom perception in IBS. Seventy-eight healthy subjects (controls) and 223 patients with IBS were enrolled in the study and phenotyped according to validated questionnaires. Sugar test was used to evaluate invivo permeability. Immunohistochemistry, western blot, and electron microscopy were used to characterize the vascular barrier. Vascular permeability was evaluated by assessing the mucosal expression of plasmalemma vesicle-associated protein-1 and vascular endothelial cadherin. Caco-2 or human umbilical vein endothelial cell monolayers were incubated with soluble mediators released by mucosal biopsies to highlight the mechanisms involved in permeability alteration. Correlation analyses have been performed among experimental and clinical data. The intestinal epithelial barrier was compromised in patients with IBS throughout the gastrointestinal tract. IBS-soluble mediators increased Caco-2 permeability via a downregulation of tight junction gene expression. Blood vessel density and vascular permeability were increased in the IBS colonic mucosa. IBS mucosal mediators increased permeability in human umbilical vein endothelial cell monolayers through the activation of protease-activated receptor-2 and histone deacetylase 11, resulting in vascular endothelial cadherin downregulation. Permeability changes correlated with intestinal and behavioral symptoms and health-related quality of life of patients with IBS. Epithelial and vascular barriers are compromised in patients with IBS and contribute to clinical manifestations.

Exogenous autoinducer-2 alleviates intestinal damage in necrotizing enterocolitis via PAR2/MMP3 signaling pathway

BackgroundImbalanced intestinal microbiota and damage to the intestinal barrier contribute to the development of necrotizing enterocolitis (NEC). Autoinducer-2 (AI-2) plays a crucial role in repairing intestinal damage and reducing inflammation. ObjectiveThis study aimed to investigate the impact of AI-2 on the expression of intestinal zonula occludens-1 (ZO-1) and occludin proteins in NEC. We evaluated its effects in vivo using NEC mice and in vitro using lipopolysaccharide (LPS)-stimulated intestinal cells. MethodsPathological changes in the intestines of neonatal mice were assessed using histological staining and scoring. Cell proliferation was measured using the cell counting kit-8 (CCK-8) assay to determine the optimal conditions for LPS and AI-2 interventions. Real-time quantitative polymerase chain reaction (RT-qPCR) was used to analyze the mRNA levels of matrix metalloproteinase-3 (MMP3), protease activated receptor-2 (PAR2), interleukin-1β (IL-1β), and IL-6. Protein levels of MMP3, PAR2, ZO-1, and occludin were evaluated using western blot, immunohistochemistry, or immunofluorescence. ResultsAI-2 alleviated NEC-induced intestinal damage (P < 0.05) and enhanced the proliferation of damaged IEC-6 cells (P < 0.05). AI-2 intervention reduced the mRNA and protein expressions of MMP3 and PAR2 in intestinal tissue and cells (P < 0.05). Additionally, it increased the protein levels of ZO-1 and occludin (P < 0.05), while reducing IL-1β and IL-6 mRNA expression (P < 0.05). ConclusionAI-2 intervention enhances the expression of tight junction proteins (ZO-1 and occludin), mitigates intestinal damage in NEC neonatal mice and IEC-6 cells, potentially by modulating PAR2 and MMP3 signaling. AI-2 holds promise as a protective intervention for NEC. AI-2 plays a crucial role in repairing intestinal damage and reducing inflammation.