Nafamostat mesylate decreases skin flap necrosis in a mouse model of type 2 diabetes by protecting the endothelial glycocalyx.

The success rate of flap tissue reconstruction has increased in recent years owing to advancements in microsurgical techniques. However, complications, such as necrosis, are still more prevalent in diabetic patients compared to non-diabetic individuals, presenting an ongoing challenge. To address this issue, many previous studies have examined vascular anastomoses dilation and stability, primarily concerning surgical techniques or drugs. In contrast, in the present study, we focused on microvascular damage of the peripheral microvessels in patients with diabetes mellitus and the preventative impact of nafamostat mesylate. Herein, we aimed to investigate the effects of hyperglycemia on glycocalyx (GCX) levels in mice with type 2 diabetes. We examined the endothelial GCX (eGCX) in skin flap tissue of 9-12-week-old type 2 diabetic mice (db/db mice) using a perforator skin flap and explored treatment with nafamostat mesylate. The growth rates were compared after 1 week. Heterotype (db/+) mice were used as the control group. Morphological examination of postoperative tissues was performed at 1, 3, 5, and 7 days post-surgery. In addition, db/db mice were treated with 30 mg/kg/day of nafamostat mesylate daily and were evaluated on postoperative day 7. Seven days after surgery, all db/db mice showed significant partial flap necrosis. Temporal observation of the skin flaps revealed a stasis-like discoloration and necrosis starting from the contralateral side of the remaining perforating branch. The control group did not exhibit flap necrosis, and the flap remained intact. In the quantitative assessment of endothelial glycans using lectins, intensity scoring showed that the eGCX in the db/db group was significantly thinner than that in the db/+ group. These results were consistent with the scanning electron microscopy findings. In contrast, treatment with nafamostat mesylate significantly improved the flap engraftment rate and suppressed eGCX injury. In conclusion, treatment with nafamostat mesylate improves the disrupted eGCX structure of skin flap tissue in db/db mice, potentially ameliorating the impaired capillary-to-venous return in the skin flap tissue.

Impact of augmented renal clearance on anticoagulant therapy in critically ill patients with coronavirus disease 2019: A retrospective cohort study.

INTRODUCTION: This study aimed to determine the impact of augmented renal clearance (ARC) on anticoagulation therapy in critically ill patients with coronavirus disease 2019 (COVID-19). METHODS: This retrospective cohort study included adult patients with severe COVID-19 with ARC who had been treated at our hospital between 2020 and 2021. We measured the estimated glomerular filtration rate calculated by the Chronic Kidney Disease Epidemiology Collaboration formula (eGFRCKD-EPI) every morning, and ARC condition was defined as eGFRCKD-EPI ≥ 130 mL/min/1.73 m2. Multivariate regression analysis with Huber-White sandwich estimator was performed to examine the association of unfractionated heparin (UH) dosage between blood test timings with activated partial thromboplastin time (APTT) compared with and without ARC. RESULTS: We identified 38 enrolled patients: seven and 31 in the ARC and non-ARC groups, respectively. In the ARC coexisting condition, a higher dose of UH, which corresponded to the total dose in 24 h from the previous day, was required to achieve the same APTT prolongation, with a significant difference (p < 0.001). CONCLUSIONS: Our study suggests that careful monitoring and consideration of higher UH doses in critically ill patients with COVID-19 is necessary because anticoagulation failure can occur during ARC.

Decreased neutrophil counts prolong inflammation in acute pancreatitis and cause inflammation spillover to distant organs.

BACKGROUND/OBJECTIVE: Acute pancreatitis is an aseptic inflammation caused by pathologically activated pancreatic enzymes and inflammatory mediators produced secondarily by neutrophils and other inflammatory cells and is one of the most difficult diseases to treat. This study aimed to investigate the role of neutrophils in pancreatitis by examining tissue dynamics. METHODS: We created a model of caerulein-induced pancreatitis in 12-week-old male granulocyte colony-stimulating factor knockout mice (G-CSF-KO) and wild-type littermate control mice (six intraperitoneal injections of caerulein [80 µg/kg body weight] at hourly intervals for 2 days). Mice were sacrificed 0, 3, 6, 12, 24, 36, 48, 72, and 168 h after caerulein administration and examined histologically. RESULTS: The survival rate after one week of caerulein administration was 100 % in the control mice, whereas it was significantly lower (10 %) in the G-CSF-KO mice. Histological examination revealed significant hemorrhage and inflammatory cell migration in the G-CSF-KO mice, indicating prolonged inflammation. CONCLUSION: Prolonged inflammation was observed in the G-CSF-KO mice. Tissue cleanup by neutrophils during the acute phase of inflammation may influence healing through the chronic phase.

Delayed diagnosis of Lemierre's syndrome in a patient with severe coronavirus disease 2019: importance of comprehensive oral and neck examination - a case report.

BACKGROUND: Given the widespread prevalence of the coronavirus disease 2019 (COVID-19), oral and neck examinations tend to be avoided in patients with suspected or confirmed COVID-19. This might delay the diagnosis of conditions such as Lemierre's syndrome, which involves symptoms resembling COVID-19-related throat manifestations. CASE PRESENTATION: A 24-year-old man without any underlying conditions was diagnosed with COVID-19 7 days before presentation. He was admitted to another hospital 1 day before presentation with severe COVID-19 and suspected bacterial pneumonia; accordingly, he was started on treatment with remdesivir and meropenem. Owing to bacteremic complications, the patient was transferred to our hospital for intensive care. On the sixth day, the patient experienced hemoptysis; further, a computed tomography (CT) scan revealed new pulmonary artery pseudoaneurysms. Successful embolization was performed to achieve hemostasis. In blood cultures conducted at the previous hospital, Fusobacterium nucleatum was isolated, suggesting a cervical origin of the infection. A neck CT scan confirmed a peritonsillar abscess and left internal jugular vein thrombus; accordingly, he was diagnosed with Lemierre's syndrome. The treatment was switched to ampicillin/sulbactam, based on the drug susceptibility results. After 6 weeks of treatment, the patient completely recovered without complications. CONCLUSION: This case highlights the significance of thorough oral and neck examinations in patients with suspected or diagnosed COVID-19 for the detection of throat and neck symptoms caused by other conditions.

Diagnostic significance of secondary bacteremia in patients with COVID-19.

OBJECTIVES: We investigated the occurrence of non-respiratory bacterial and fungal secondary infections, causative organisms, impact on clinical outcomes, and association between the secondary pathogens and mortality in hospitalized patients with coronavirus disease 2019 (COVID-19). METHODS: This was a retrospective cohort study that included data from inpatients with COVID-19 from multiple centers participating in the Japan COVID-19 Taskforce (April 2020 to May 2021). We obtained demographic, epidemiological, and microbiological data throughout the course of hospitalization and analyzed the cases of COVID-19 complicated by non-respiratory bacterial infections. RESULTS: Of the 1914 patients included, non-respiratory bacterial infections with COVID-19 were diagnosed in 81 patients (4.2%). Of these, 59 (3.1%) were secondary infections. Bacteremia was the most frequent bacterial infection, occurring in 33 cases (55.9%), followed by urinary tract infections in 16 cases (27.1%). Staphylococcus epidermidis was the most common causative organism of bacteremia. Patients with COVID-19 with non-respiratory secondary bacterial infections had significantly higher mortality, and a multivariate logistic regression analysis demonstrated that those with bacteremia (aOdds Ratio = 15.3 [5.97-39.1]) were at higher risk of death. Multivariate logistic regression analysis showed that age, male sex, use of steroids to treat COVID-19, and intensive care unit admission increased the risk for nosocomial bacteremia. CONCLUSIONS: Secondary bacteremia is an important complication that may lead to poor prognosis in cases with COVID-19. An appropriate medical management strategy must be established, especially for patients with concomitant predisposing factors.

Impact of respiratory bacterial infections on mortality in Japanese patients with COVID-19: a retrospective cohort study.

BACKGROUND: Although cases of respiratory bacterial infections associated with coronavirus disease 2019 (COVID-19) have often been reported, their impact on the clinical course remains unclear. Herein, we evaluated and analyzed the complication rates of bacterial infections, causative organisms, patient backgrounds, and clinical outcome in Japanese patients with COVID-19. METHODS: We performed a retrospective cohort study that included inpatients with COVID-19 from multiple centers participating in the Japan COVID-19 Taskforce (April 2020 to May 2021) and obtained demographic, epidemiological, and microbiological results and the clinical course and analyzed the cases of COVID-19 complicated by respiratory bacterial infections. RESULTS: Of the 1,863 patients with COVID-19 included in the analysis, 140 (7.5%) had respiratory bacterial infections. Community-acquired co-infection at COVID-19 diagnosis was uncommon (55/1,863, 3.0%) and was mainly caused by Staphylococcus aureus, Klebsiella pneumoniae and Streptococcus pneumoniae. Hospital-acquired bacterial secondary infections, mostly caused by Staphylococcus aureus, Pseudomonas aeruginosa, and Stenotrophomonas maltophilia, were diagnosed in 86 patients (4.6%). Severity-associated comorbidities were frequently observed in hospital-acquired secondary infection cases, including hypertension, diabetes, and chronic kidney disease. The study results suggest that the neutrophil-lymphocyte ratio (> 5.28) may be useful in diagnosing complications of respiratory bacterial infections. COVID-19 patients with community-acquired or hospital-acquired secondary infections had significantly increased mortality. CONCLUSIONS: Respiratory bacterial co-infections and secondary infections are uncommon in patients with COVID-19 but may worsen outcomes. Assessment of bacterial complications is important in hospitalized patients with COVID-19, and the study findings are meaningful for the appropriate use of antimicrobial agents and management strategies.

Synergy by Ristocetin and CXCL12 in Human Platelet Activation: Divergent Regulation by Rho/Rho-Kinase and Rac.

CXCL12, belonging to the CXC chemokine family, is a weak agonist of platelet aggregation. We previously reported that the combination of CXCL12 and collagen at low doses synergistically activates platelets via not CXCR7 but CXCR4, a specific receptor for CXCL12 on the plasma membrane. Recently, we reported that not Rho/Rho kinase, but Rac is involved in the platelet aggregation induced by this combination. Ristocetin is an activator of the von Willebrand factor that interacts with glycoprotein (GP) Ib/IX/V, which generates thromboxane A2 via phospholipase A2 activation, resulting in the release of the soluble CD40 ligand (sCD40L) from human platelets. In the present study, we investigated the effects of a combination of ristocetin and CXCL12 at low doses on human platelet activation and its underlying mechanisms. Simultaneous stimulation with ristocetin and CXCL12 at subthreshold doses synergistically induce platelet aggregation. A monoclonal antibody against not CXCR7 but CXCR4 suppressed platelet aggregation induced by the combination of ristocetin and CXCL12 at low doses. This combination induces a transient increase in the levels of both GTP-binding Rho and Rac, followed by an increase in phosphorylated cofilin. The ristocetin and CXCL12-induced platelet aggregation as well as the sCD40L release were remarkably enhanced by Y27362, an inhibitor of Rho-kinase, but reduced by NSC23766, an inhibitor of the Rac-guanine nucleotide exchange factor interaction. These results strongly suggest that the combination of ristocetin and CXCL12 at low doses synergistically induces human platelet activation via Rac and that this activation is negatively regulated by the simultaneous activation of Rho/Rho-kinase.

Recombinant Antithrombin Attenuates Acute Respiratory Distress Syndrome in Experimental Endotoxemia.

Sepsis-induced endothelial acute respiratory distress syndrome is related to microvascular endothelial dysfunction caused by endothelial glycocalyx disruption. Recently, recombinant antithrombin (rAT) was reported to protect the endothelial glycocalyx from septic vasculitis; however, the underlying mechanism remains unknown. Here, we investigated the effect of rAT administration on vascular endothelial injury under endotoxemia. Lipopolysaccharide (LPS; 20 mg/kg) was injected intraperitoneally into 10-week-old male C57BL/6 mice, and saline or rAT was administered intraperitoneally at 3 and 24 hours after LPS administration. Subsequently, serum and/or pulmonary tissues were examined for inflammation and cell proliferation and differentiation by histologic, ultrastructural, and microarray analyses. The survival rate was significantly higher in rAT-treated mice than in control mice 48 hours after LPS injection (75% versus 20%; P < 0.05). Serum interleukin-1ß was increased but to a lesser extent in response to LPS injection in rAT-treated mice than in control mice. Lectin staining and ultrastructural studies showed a notable attenuation of injury to the endothelial glycocalyx after rAT treatment. Microarray analysis further showed an up-regulation of gene sets corresponding to DNA repair, such as genes involved in DNA helicase activity, regulation of telomere maintenance, DNA-dependent ATPase activity, and ciliary plasm, after rAT treatment. Thus, rAT treatment may promote DNA repair, attenuate inflammation, and promote ciliogenesis, thereby attenuating the acute respiratory distress syndrome caused by endothelial injury.

Amyloid ß protein negatively regulates human platelet activation induced by thrombin receptor-activating protein.

Amyloid ß protein deposition in cerebral vessels, a characteristic of Alzheimer's disease, is a risk factor for intracerebral hemorrhage. Amyloid ß protein directly modulates human platelet function; however, the exact mechanism of action is unclear. Therefore, we investigated the effects of amyloid ß protein on human platelet activation using an aggregometer with laser scattering. Amyloid ß protein decreased platelet aggregation induced by thrombin receptor-activating protein, but not by collagen and ADP. Amyloid ß protein also suppressed platelet aggregation induced by SCP0237 and A3227. Platelet-derived growth factor-AB secretion and phosphorylated-heat shock protein 27 release by thrombin receptor-activating protein were inhibited by amyloid ß protein. Additionally, thrombin receptor-activating protein-induced phosphorylation of JNK and p38 MAP kinase was reduced by amyloid ß protein. Collectively, our results strongly suggest that amyloid ß protein negatively regulates protease-activated receptor-elicited human platelet activation. These findings may indicate a cause of intracerebral hemorrhage due to amyloid ß protein.

Relationship between the Responsiveness of Amyloid ß Protein to Platelet Activation by TRAP Stimulation and Brain Atrophy in Patients with Diabetes Mellitus.

Type 2 DM is a risk factor for dementia, including Alzheimer's disease (AD), and is associated with brain atrophy. Amyloid ß protein (Aß) deposition in the brain parenchyma is implicated in the neurodegeneration that occurs in AD. Platelets, known as abundant storage of Aß, are recognized to play important roles in the onset and progression of AD. We recently showed that Aß negatively regulates platelet activation induced by thrombin receptor-activating protein (TRAP) in healthy people. In the present study, we investigated the effects of Aß on the TRAP-stimulated platelet activation in DM patients, and the relationship between the individual responsiveness to Aß and quantitative findings of MRI, the volume of white matter hyperintensity (WMH)/intracranial volume (IC) and the volume of parenchyma (PAR)/IC. In some DM patients, Aß reduced platelet aggregation induced by TRAP, while in others it was unchanged or rather enhanced. The TRAP-induced levels of phosphorylated-Akt and phosphorylated-HSP27, the levels of PDGF-AB and the released phosphorylated-HSP27 correlated with the degree of platelet aggregability. The individual levels of not WMH/IC but PAR/IC was correlated with those of TRAP-stimulated PDGF-AB release. Collectively, our results suggest that the reactivity of TRAP-stimulated platelet activation to Aß differs in DM patients from healthy people. The anti-suppressive feature of platelet activation to Aß might be protective for brain atrophy in DM patients.

Vascular endothelial injury exacerbates coronavirus disease 2019: The role of endothelial glycocalyx protection.

The potential for a rapid increase in severity is among the most frightening aspects of severe acute respiratory syndrome coronavirus 2 infection. Evidence increasingly suggests that the symptoms of coronavirus disease-2019 (COVID-19)-related acute respiratory distress syndrome (ARDS) differ from those of classic ARDS. Recently, the severity of COVID-19 has been attributed to a systemic, thrombotic, and inflammatory disease that damages not only the lungs but also multiple organs, including the heart, brain, toes, and liver. This systemic form of COVID-19 may be due to inflammation and vascular endothelial cell injury. The vascular endothelial glycocalyx comprises glycoproteins and plays an important role in systemic capillary homeostasis maintenance. The glycocalyx covers the entire vascular endothelium, and its thickness varies among organs. The endothelial glycocalyx is very thin in the pulmonary capillaries, where it is affected by gaseous exchange with the alveoli and the low intravascular pressure in the pulmonary circulation. Despite the clearly important roles of the glycocalyx in vascular endothelial injury, thrombosis, vasculitis, and inflammation, the link between this structure and vascular endothelial cell dysfunction in COVID-19 remains unclear. In this prospective review, we summarize the importance of the glycocalyx and its potential as a therapeutic target in cases of systemic COVID-19.

Possible involvement of Syndecan-1 in the state of COVID-19 related to endothelial injury.

BACKGROUND: The coronavirus infection 2019 (COVID-19) is associated with microvascular endothelial injury. Here, we report that syndecan-1, a component of endothelial glycocalyx, may reflect the disease state of COVID-19 related to endothelial injury. CASE PRESENTATION: A patient with COVID-19 was transferred to the intensive care unit of our hospital. Computed tomography of the chest showed bilateral ground glass opacities, which was diagnosed as acute respiratory syndrome. The PaO2/FIO2 ratio gradually increased from 158 on hospitalization to 300 on Day 11, on which day the ventilator was withdrawn. However, serum syndecan-1 (SDC-1) level gradually decreased from 400.5 ng/ml at hospitalization to 165.1 ng/ml on Day 5. On Day 6, serum SDC-1 level increased to 612.9 ng/ml owing to a systemic thrombosis with an increase in D-dimer. Serum SDC-1 level then decreased until 206.0 ng/ml on Day 11 after a decrease in D-dimer. The patient was transferred to another hospital on Day 21 after hospitalization. CONCLUSIONS: In this case report, changes in serum SDC-1 level closely reflected the change in disease condition in a patient with COVID-19. Serum SDC-1 may be a useful biomarker for monitoring the disease state of critically ill patients with COVID-19.

Intraocular penetration of liposomal amphotericin B after intravenous injection in inflamed human eyes.

PURPOSE: To determine the intraocular penetration of amphotericin B (AMPH-B) after an intravenously injection of liposomal amphotericin B (L-AMB) in inflamed human eyes. METHODS: Seven eyes of 5 patients with fungal eye diseases (endophthalmitis in 6 eyes and keratitis in 1 eye) were treated with intravenous injections of 100-250 mg/day of L-AMB. Samples of blood, corneal button, aqueous humor, and vitreous humor were collected and assessed for AMPH-B. RESULTS: The AMPH-B level in the cornea (604.0 µg/g) of the case with fungal keratitis exceeded the minimum inhibitory concentration. However, the levels in the aqueous and vitreous humors of the cases with fungal endophthalmitis were lower, e.g., 0.02 ± 0.01 µg/ml (0.09% of serum level) in the aqueous humor and 0.05 ± 0.08 µg/ml (0.17% of serum level) in the vitreous humor. CONCLUSIONS: The AMPH-B levels administered intravenously were very low in the aqueous and vitreous humors. Our findings indicate that intravenous L-AMB can be considered only for patients with mild endogenous fungal endophthalmitis, e.g., isolated chorioretinitis without vitreous extensions.

Validity and risk factor analysis for helicopter emergency medical services in Japan: a pilot study.

BACKGROUND: Some emergency departments use triage scales, such as the Canadian Triage and Acuity Scale and Japan Urgent Stroke Triage Score, to detect life-threatening situations. However, these protocols have not been used for aeromedical services. Therefore, we investigated the factors predicting these life-threatening situations in aeromedical services as a pilot study for establishing the protocol. METHOD: We retrospectively evaluated helicopter emergency medical service cases from 1 April 2015 to 31 March 2020 at Gifu University Hospital using the mission records. We only evaluated cases dealing with suggested internal medicine issues. We excluded cases influenced by external factors such as trauma or cases that included hospital-to-hospital transportation, focusing only on prehospital care. We evaluated the validity of the medical emergencies based on the needs for emergency interventions and hospital admission and of the suggested diagnoses and associated risk factors. RESULT: A total of 451 cases were suitable for inclusion in the study. In the analysis for all emergency calls, 235 (52.11%) cases needed emergency intervention and 300 (64.4%) required hospital admission. The suggested diagnosis was valid for 261 (57.87%) cases. After the first assessment by emergency medical technicians, 75 cases were removed. Analysis after this first assessment found that 52.31% cases required emergency intervention, 70.26% needed admission, and the suggested diagnosis was valid for 69.41% of cases. In the analysis of emergency calls, the multivariate analysis of some key variables identified age, playing sports, and gasping as risk factors for emergency intervention. Hospital admission risk factors included being age only. The suggested diagnosis was valid only for sports situations. In the analysis after the first assessment by an emergency medical technician, risk factors for emergency intervention included being age being male, playing sports, and gasping, and those for hospital admission was being age, being male, and experiencing stroke symptoms and/or disturbance of consciousness. The suggested diagnosis was valid only for sports situations. CONCLUSION: Some 'second' keywords/phrases predict medical emergencies. Therefore, the dispatch commander should gather these keyword/phrases to assess.

Neutrophil Elastase Damages the Pulmonary Endothelial Glycocalyx in Lipopolysaccharide-Induced Experimental Endotoxemia.

Neutrophil elastase (NE) is necessary for effective sterilization of phagocytosed bacterial and fungal pathogens; however, NE increases alveolocapillary permeability and induces proinflammatory cytokine production in sepsis-induced acute respiratory distress syndrome. Under septic conditions, the pulmonary endothelial glycocalyx covering on the healthy endothelium surface is injured, but the contribution of NE to this injury remains unknown. Our aim was to examine whether NE-induced pulmonary endothelial injury is associated with endotoxemia. Lipopolysaccharide (LPS; 20 mg/kg) was injected intraperitoneally into 9- to 12-week-old granulocyte colony-stimulating factor knockout (G-CSFKO) mice, which harbor few neutrophils, and littermate control mice; in a second assay, mice were injected with the NE-inhibitor sivelestat (0.2 mg/kg) at 3, 6, 9, and 12 hours after LPS administration. Subsequently, vascular endothelial injury was evaluated through ultrastructural analysis. At 48 hours after LPS injection, survival rate was more than threefold higher among G-CSFKO than control mice, and degradation of both thrombomodulin and syndecan-1 was markedly attenuated in G-CSFKO compared with control mice. Ultrastructural analysis revealed attenuated vascular endothelial injury and clear preservation of the endothelial glycocalyx in G-CSFKO mice. Moreover, after LPS exposure, survival rate was approximately ninefold higher among sivelestat-injected mice than control mice, and sivelestat treatment potently preserved vascular endothelial structures and the endothelial glycocalyx. In conclusion, NE is associated with pulmonary endothelial injury under LPS-induced endotoxemic conditions.

Multiple trauma including pelvic fracture with multiple arterial embolization: an autopsy case report.

BACKGROUND: Pelvic fracture with high energy trauma has a high mortality rate, especially in men. In addition, severe multiple trauma, major hemorrhage, and administration of red blood cells predict mortality in elderly patients with pelvic fracture. We herein report a rare case in which multiple arterial embolization occurred after pelvic fracture. CASE PRESENTATION: An 83-year-old male cyclist was transported to our hospital after being struck by a car. On arrival, he was diagnosed with multiple trauma, including rib fractures with hemothorax, lumbar fractures of the transverse process, and injuries in the right acetabulum, left adrenal gland, and liver. He underwent massive transfusion and transcatheter arterial embolization due to extravasation from the right superior gluteal artery and left adrenal gland. On the second day, owing to right lower leg ischemia, serum creatinine kinase and myoglobin levels were markedly elevated from the reference value; hence, a right above-knee amputation was performed 12 h after the accident. However, both protein levels remained high after amputation, resulting in acute renal injury, which was treated via hemodiafiltration on hospital day 3. In addition, sustained low efficiency hemodialysis and plasma exchange were performed on hospital day 4. Despite these treatments, the patient's hemodynamics did not improve, and he died on hospital day 8. The autopsy revealed necropsy of the iliopsoas muscles and the digestive tract. CONCLUSIONS: The causes of the patient's death were considered to be persistent rhabdomyolysis and severe hypotension due to iliopsoas necrosis and peritonitis due to digestive tract necrosis. Multiple arterial embolization caused by consumption coagulopathy associated with multiple trauma may account for severe outcomes in this case.

Olive polyphenol reduces the collagen-elicited release of phosphorylated HSP27 from human platelets.

Hydroxytyrosol (HT) and oleuropein (OLE) are natural polyphenols found in extra virgin olive oil. Accumulating evidence indicates that ingestion of olive oil contributes to reduce the risk of cardiovascular diseases and stroke. It has been reported that HT and OLE inhibit human platelet aggregation. We have shown that collagen induces the phosphorylation of heat shock protein 27 (HSP27) in human platelets, resulting in the release of HSP27, an extracellular pro-inflammatory agent. In this study, we investigated the effects of HT and OLE on the collagen-stimulated human platelet activation. The PDGF-AB secretion and the soluble CD40 ligand (sCD40L) release by collagen were reduced by HT or OLE. HT and OLE significantly suppressed the phosphorylation of HSP27 and the release of phosphorylated-HSP27. These findings suggest that olive polyphenol reduces the collagen-stimulated phosphorylation of HSP27 in human platelets and the release. Our results may provide a novel anti- inflammatory effect of olive polyphenol.

Rac Regulates the TRAP-Induced Release of Phosphorylated-HSP27 from Human Platelets via p38 MAP Kinase but Not JNK.

BACKGROUND/AIMS: Thrombin induces the activation of human platelets through protease-activated receptor (PAR) 1 and PAR4, and Rac, a member of the Rho family of small GTPases, is implicated in PAR activation. We previously reported that phosphorylated-heat shock protein 27 (HSP27) is released from the thrombin receptor-activating peptide (TRAP)-stimulated platelets of diabetic patients. In the present study, we investigated the role of Rac in the TRAP-elicited release of phosphorylated-HSP27 from human platelets. METHODS: Platelet aggregation was measured using an aggregometer with laser scattering. Protein phosphorylation was analyzed by Western blotting. The levels of phosphorylated-HSP27 and platelet-derived growth factor-AB (PDGF-AB) were measured by enzyme-linked immunosorbent assays. RESULTS: NSC23766, an inhibitor of Rac-guanine nucleotide exchange factor interaction, suppressed the TRAP-elicited release of phosphorylated-HSP27 as well as platelet aggregation. The TRAP-induced phosphorylation of HSP27, p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK) was attenuated by NSC23766. SB203580, a p38 MAPK inhibitor, but not SP600125, a JNK inhibitor, suppressed the release of phosphorylated-HSP27 in addition to HSP27 phosphorylation. On the other hand, both SB203580 and SP600125 reduced the TRAP-stimulated secretion of PDGF-AB. CONCLUSION: Our results strongly suggest that Rac acts as a positive regulator of the PAR-elicited release of phosphorylated-HSP27 from human platelets via p38 MAPK but not JNK.

Three-dimensional ultrastructure of capillary endothelial glycocalyx under normal and experimental endotoxemic conditions.

BACKGROUND: Sugar-protein glycocalyx coats healthy endothelium, but its ultrastructure is not well described. Our aim was to determine the three-dimensional ultrastructure of capillary endothelial glycocalyx in the heart, kidney, and liver, where capillaries are, respectively, continuous, fenestrated, and sinusoidal. METHODS: Tissue samples were processed with lanthanum-containing alkaline fixative, which preserves the structure of glycocalyx. RESULTS: Scanning and transmission electron microscopy revealed that the endothelial glycocalyx layer in continuous and fenestrated capillaries was substantially thicker than in sinusoids. In the heart, the endothelial glycocalyx presented as moss- or broccoli-like and covered the entire luminal endothelial cell surface. In the kidney, the glycocalyx appeared to nearly occlude the endothelial pores of the fenestrated capillaries and was also present on the surface of the renal podocytes. In sinusoids of the liver, glycocalyx covered not only the luminal side but also the opposite side, facing the space of Disse. In a mouse lipopolysaccharide-induced experimental endotoxemia model, the capillary endothelial glycocalyx was severely disrupted; that is, it appeared to be peeling off the cells and clumping. Serum concentrations of syndecan-1, a marker of glycocalyx damage, were significantly increased 24 h after administration of lipopolysaccharide. CONCLUSIONS: In the present study, we visualized the three-dimensional ultrastructure of endothelial glycocalyx in healthy continuous, fenestrated, and sinusoidal capillaries, and we also showed their disruption under experimental endotoxemic conditions. The latter may provide a morphological basis for the microvascular endothelial dysfunction associated with septic injury to organs.

Postinfarction Cardiac Remodeling Proceeds Normally in Granulocyte Colony-Stimulating Factor Knockout Mice.

Treatment with granulocyte colony-stimulating factor (G-CSF) reportedly mitigates postinfarction cardiac remodeling and dysfunction. We herein examined the effects of G-CSF knockout (G-CSF-KO) on the postinfarction remodeling process in the hearts of mice. Unexpectedly, the acute infarct size 24 hours after ligation was similar in the two groups. At the chronic stage (4 weeks later), there was no difference in the left ventricular dimension, left ventricular function, or histological findings, including vascular density, between the two groups. In addition, expression of vascular endothelial growth factor (VEGF) was markedly up-regulated in hearts from G-CSF-KO mice, compared with wild-type mice. Microarray failed in detecting up-regulation of VEGF mRNA, whereas G-CSF administration significantly decreased myocardial VEGF expression in mice, indicating that G-CSF post-transcriptionally down-regulates VEGF expression. When G-CSF-KO mice were treated with an anti-VEGF antibody (bevacizumab), cardiac remodeling was significantly aggravated, with thinning of the infarct wall and reduction of the cellular component, including blood vessels. In the granulation tissue of bevacizumab-treated hearts 4 days after infarction, vascular development was scarce, with reduced cell proliferation and increased apoptosis, which likely contributed to the infarct wall thinning and the resultant increase in wall stress and cardiac remodeling at the chronic stage. In conclusion, overexpression of VEGF may compensate for the G-CSF deficit through preservation of cellular components, including blood vessels, in the postinfarction heart.