Intermittent rolling is a defect of the extravasation cascade caused by Myosin1e-deficiency in neutrophils.

Neutrophil extravasation is a migratory event in response to inflammation that depends on cytoskeletal dynamics regulated by myosins. Myosin-1e (Myo1e) is a long-tailed class-I myosin that has not yet been studied in the context of neutrophil-endothelial interactions and neutrophil extravasation. Intravital microscopy of TNFα-inflamed cremaster muscles in Myo1e-deficient mice revealed that Myo1e is required for efficient neutrophil extravasation. Specifically, Myo1e deficiency caused increased rolling velocity, decreased firm adhesion, aberrant crawling, and strongly reduced transmigration. Interestingly, we observed a striking discontinuous rolling behavior termed "intermittent rolling," during which Myo1e-deficient neutrophils showed alternating rolling and jumping movements. Surprisingly, chimeric mice revealed that these effects were due to Myo1e deficiency in leukocytes. Vascular permeability was not significantly altered in Myo1e KO mice. Myo1e-deficient neutrophils showed diminished arrest, spreading, uropod formation, and chemotaxis due to defective actin polymerization and integrin activation. In conclusion, Myo1e critically regulates adhesive interactions of neutrophils with the vascular endothelium and neutrophil extravasation. Myo1e may therefore be an interesting target in chronic inflammatory diseases characterized by excessive neutrophil recruitment.

Homoectoine Protects Against Colitis by Preventing a Claudin Switch in Epithelial Tight Junctions.

BACKGROUND: Inflammatory bowel diseases (IBD) are multifactorial disorders affecting millions of people worldwide with alarmingly increasing incidences every year. Dysfunction of the intestinal epithelial barrier is associated with IBD pathogenesis, and therapies include anti-inflammatory drugs that enhance intestinal barrier function. However, these drugs often have adverse side effects thus warranting the search for alternatives. Compatible solutes such as bacterial ectoines stabilize cell membranes and proteins. AIM: To unravel whether ectoine (1,4,5,6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid) and homoectoine (4,5,6,7-tetrahydro-2-methyl-1H-(1,3)-diazepine-4-carboxylic acid), a synthetic derivative of ectoine, have beneficial effects during dextran sulfate sodium (DSS)-induced colitis in mice. METHODS/RESULTS: We found that the disease activity index was significantly reduced by both ectoines. DSS-induced edema formation, epithelial permeability, leukocyte recruitment and tissue damage were reduced by ectoine and homoectoine, with the latter having stronger effects. Interestingly, the claudin switch usually observed during colitis (decreased expression of claudin-1 and increased expression of the leaky claudin-2) was completely prevented by homoectoine, whereas ectoine only reduced claudin-2 expression. Concomitantly, only homoectoine ameliorated the drop in transepithelial electrical resistance induced by IFN-γ and TNF-α in Caco-2 cells. Both ectoines inhibited loss of ZO-1 and occludin and prevented IFN-γ/TNF-α-induced increased paracellular flux of 4 kDa FITC-dextran in vitro. Moreover, both ectoines reduced expression of pro-inflammatory cytokines and oxidative stress during colitis. CONCLUSION: While both ectoine and homoectoine have protective effects on the epithelial barrier during inflammation, only homoectoine completely prevented the inflammatory claudin switch in tight junctions. Thus, homoectoine may serve as diet supplement in IBD patients to reach or extend remission.

Comparative effects of nitric oxide dependent and independent vasodilation on impaired hindlimb revascularization in eNOS-/- mice.

Ischemia due to vascular occlusion induces vasodilation as an initial response, followed by arteriogenesis or angiogenesis. Vasodilation through nitric oxide (NO) independent and dependent mechanisms may be sufficient to restore the altered neovascularization in pathological situations where the NO is altered. Using a posterior limb claudication model to evaluate ischemia-induced revascularization in eNOS-/- mice, we compared the effects of sodium nitrite, a NO-dependent vasodilator, and prazocin, an alpha-adrenergic blocker and NO-independent vasodilator, on hindlimb revascularization. We evaluated the blood flow of the hindlimbs, NO and nitrites metabolites, the expression of tissue endothelial cell markers and proangiogenic factors, as well as the gait locomotion. Our results suggest that the use of a peripheral vasodilator can substitute the initial absence of NO as an endogenous vasodilator. However, final resolution of the ischemic process requires a NO-mediated pathway, which through changes in vascular hemodynamics, promotes the generation of angiogenic messengers facilitating the functional recovery of the damaged limb.

Role of sodium/glucose cotransporter inhibition on a rat model of angiotensin II-dependent kidney damage.

BACKGROUND: Renal proximal tubular sodium and glucose reabsorption are regulated by the sodium-glucose cotransporter (SGLT2). Changes in this transporter can play a role in hyperglycaemia and reactive oxygen species (ROS) production. We demonstrated increased glucose absorption in proximal tubule membrane vesicles and increased expression of SGLT2 in hypertensive rats. Here we investigated Angiotensin II (Ang II) -dependent SGLT2 expression induction and the role of SGLT2 induction in the development of Ang II-dependent kidney damage. The aim of this study was to determine whether SGLT2 induction by Ang II is associated with Ang II-dependent kidney damage. We propose the following objectives a) to demonstrate that Ang II induces SGLT2 expression and b) to demonstrate that prevention of SGLT2 expression and activity prevent Ang II-induced kidney damage. METHODS: We used chronic Ang II infusion as a model of kidney damage in male Wistar rats and evaluated systolic blood pressure by telemetric methods. SGLT2 mRNA and protein expression were evaluated by PCR and immunoblotting. SGLT2 activity was evaluated in brush border membrane vesicles by measuring glucose uptake. ROS production was measured by confocal microscopy. The glomerular filtration rate (GFR) was evaluated by the inulin excretion method, and urinary protein excretion was evaluated by the Bradford method. Biological parameter evaluations were performed, after two weeks of infusion of Ang II. We compared the effects of Angiotensin II (AT1) receptor blockade by Losartan and SGLT2 inhibition by Empagliflozin both as monotherapy treatments and in combination on the development of kidney damage. RESULTS: Chronic Ang II infusion led to a blood pressure elevation and increased SGLT2 mRNA expression and activity as well as kidney damage, as reflected by increased ROS production, decreased GFR and increased urinary protein excretion. AT1 receptor blockade prevented all these changes. By contrast, SGLT2 inhibition did not affect blood pressure and had a small effect on kidney damage. However, the combination of both drugs resulted in the potentiation of the effects observed by AT1 receptor blockade alone. CONCLUSIONS: We suggest that Ang II-dependent increased SGLT2 induction is one mechanism by which Ang II induces kidney damage.

Androgens Mediate ß-adrenergic Vasorelaxation Impairment Using Adenylyl Cyclase.

Cardiovascular disease development has been associated with sex differences, suggesting that sex hormones are implicated in vascular function and development of hypertension. Vascular tone comparison at different stages of rat growth represents a good model to study testosterone-related vascular response. We explored the role of testosterone in modulation of age-dependent impaired ß-adrenergic vasodilation. The 3-week-old male Sprague-Dawley rats were sorted in 3-week-old rats without any manipulation and 3-week-old rats treated with testosterone. The 9-week-old rats were randomly grouped into 9-week-old rats without any manipulation (sham), 9-week-old rats that underwent gonadectomy (9-week-old castrated), and 9-week-old castrated treated with testosterone replacement therapy (9-week-old castrated + testosterone). Vascular relaxation was evaluated in aortic rings. ß-adrenergic receptor protein expression, cyclic adenosine monophosphate production, testosterone levels, and adenylyl cyclase (AC) gene expression were assessed. Testosterone levels were low in 3-week-old and 9-week-old castrated rats compared with 9-week-old sham rats. Testosterone replacement raised these levels in 3-week-old and 9-week-old castrated rats similar to those of 9-week-old sham rats. SQ22536, the AC inhibitor, prevented isoproterenol-induced relaxation in aortic rings from 3-week-old and 9-week-old castrated rats. The ß-adrenergic receptor protein expression was similar in all experimental groups. AC mRNA and protein expression and cyclic adenosine monophosphate levels were elevated in 3-week-old and 9-week-old castrated rats compared with 3-week-old + testosterone, 9-week-old sham, and 9-week-old castrated + testosterone rats. In conclusion, we demonstrated that age maturation was associated with vascular relaxation impairment. Variations in testosterone levels and reduced AC expression may be responsible for this altered vascular function.

Cxcl1 monomer-dimer equilibrium controls neutrophil extravasation.

The chemokine Cxcl1 plays a crucial role in recruiting neutrophils in response to infection. The early events in chemokine-mediated neutrophil extravasation involve a sequence of highly orchestrated steps including rolling, adhesion, arrest, and diapedesis. Cxcl1 function is determined by its properties of reversible monomer-dimer equilibrium and binding to Cxcr2 and glycosaminoglycans. Here, we characterized how these properties orchestrate extravasation using intravital microscopy of the cremaster. Compared to WT Cxcl1, which exists as both a monomer and a dimer, the trapped dimer caused faster rolling, less adhesion, and less extravasation. Whole-mount immunofluorescence of the cremaster and arrest assays confirmed these data. Moreover, the Cxcl1 dimer showed impaired LFA-1-mediated neutrophil arrest that could be attributed to impaired Cxcr2-mediated ERK signaling. We conclude that Cxcl1 monomer-dimer equilibrium and potent Cxcr2 activity of the monomer together coordinate the early events in neutrophil recruitment.

Correlation between levels of circulating adipokines and adiponectin/resistin index with carotid intima-media thickness in hypertensive type 2 diabetic patients.

BACKGROUND: Hypoadiponectinemia and hyperresistinemia are associated with cardiovascular disease. The increase in the carotid intima-media thickness (CIMT) assessed by B-mode ultrasound has been directly associated with increased risk of myocardial infarction and stroke. OBJECTIVE: To evaluate the correlation between adipokine levels with CIMT in hypertensive type 2 diabetic patients. METHODS: Serum levels of adiponectin and resistin levels were measured by ELISA in 30 type 2 diabetic patients with never-treated hypertension and in age-matched healthy controls. The CIMT (B-mode color imaging of extracranial carotid arteries using high-resolution ultrasound) was also obtained. The relationship between adipokine levels and the adiponectin/resistin index with the CIMT was assessed by the Pearson correlation coefficient test. RESULTS: Adiponectin was lower (p < 0.05), and resistin higher (p < 0.01) in patients than in controls, CIMT correlated positively with resistin (R = 0.45, p < 0.02) and the adiponectin/resistin index (R = 0.58, p < 0.001), but not with adiponectin levels (r = -0.11, p > 0.1) in patients. Whereas only adiponectin levels correlated - negatively - with CIMT (r = -0.39, p < 0.02) in controls. CONCLUSION: Our results shown that the adiponectin/resistin index seems to be more strongly associated with atherosclerosis than adipokine levels, and may be used as a reliable marker of cardiovascular risk in type 2 diabetic hypertensive patients.

T cell functions and organ infiltration by leukemic T cells require cortactin.

T cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy that is still fatal in many cases. T cell blasts are characterized by hyperactivation and strong proliferative and migratory capacities. The chemokine receptor CXCR4 is involved in mediating malignant T cell properties, and cortactin has been shown to control CXCR4 surface localization in T-ALL cells. We have previously shown that cortactin overexpression is correlated with organ infiltration and relapse in B-ALL. However, the role of cortactin in T cell biology and T-ALL remains elusive. Here, we analyzed the functional relevance of cortactin for T cell activation and migration and the implications for T-ALL development. We found that cortactin is upregulated in response to T cell receptor engagement and recruited to the immune synapse in normal T cells. Loss of cortactin caused reduced IL-2 production and proliferation. Cortactin-depleted T cells showed defects in immune synapse formation and migrated less due to impaired actin polymerization in response to T cell receptor and CXCR4 stimulation. Leukemic T cells expressed much higher levels of cortactin compared to normal T cells that correlated with greater migratory capacity. Xenotransplantation assays in NSG mice revealed that cortactin-depleted human leukemic T cells colonized the bone marrow significantly less and failed to infiltrate the central nervous system, suggesting that cortactin overexpression drives organ infiltration, which is a major complication of T-ALL relapse. Thus, cortactin could serve as a potential therapeutic target for T-ALL and other pathologies involving aberrant T cell responses.

Cyclooxygenase-2 and kidney failure.

Cyclooxygenase (COX)-dependent prostaglandins are necessary for normal kidney function. These prostaglandins are associated with inflammation, maintenance of sodium and water homeostasis, control of renin release, renal vasodilation, vasoconstriction attenuation, and prenatal renal development. COX-2 expression is regulated by the renin-angiotensin system, glucocorticoids or mineralcorticoids, and aldosterone, supporting a role for COX-2 in kidney function. Indeed, COX-2 mRNA and protein levels as well as enzyme activity are increased, along with PGE2, during kidney failure. In addition, changes in COX-2 expression are associated with increased blood pressure, urinary volume, sodium and protein and decreased urinary osmolarity. Intrarenal mechanisms such as angiotensin II (Ang II) production, increased sodium delivery, glomerular hypertension, and renal tubular inflammation have been suggested to be responsible for the increase in COX-2 expression. Although, specific COX-2 pharmacological inhibition has been related to the prevention of kidney damage, clinical studies have reported that COX-2 inhibition may cause side effects such as edema or a modest elevation in blood pressure and could possibly interfere with antihypertensive drugs and increase the risk of cardiovascular complications. Thus, administration of COX-2 inhibitors requires caution, especially in the presence of underlying cardiovascular disease.

Beneficial effect of combination therapy using an angiotensin-converting enzyme inhibitor plus verapamil on circulating resistin levels in hypertensive patients with type 2 diabetes.

BACKGROUND: Resistin levels are strongly correlated with insulin resistance and vascular inflammation. Type 2 diabetic and hypertensive patients have higher circulating levels of resistin, which is associated with endothelial dysfunction. OBJECTIVE: To compare the effect of trandolapril (T) and its fixed-dose combination with verapamil (FDTV) on resistin levels in hypertensive, type-2 diabetic patients. METHODS: Forty type-2 diabetic patients with never-treated hypertension were randomly assigned to two groups. One group received FDTV 2 mg/180 mg once per day; the other group received T 2 mg once per day. Study drugs were administered for three months in both groups. Resistin levels were measured using ELISA at the beginning of the study and at study end. Patients were evaluated monthly for blood pressure, fasting serum glucose levels and adverse events. Statistical analysis was performed using ANOVA. RESULTS: All patients experienced a significant reduction in blood pressure. Both therapeutic regimens reduced resistin levels; however, FDTV treatment resulted in a greater decrease in resistin levels (mean [± SD] 25.5±13 ng/mL to 17.2±10 ng/mL) when compared with T treatment (22.4±12 ng/mL to 18.5±8 ng/mL) (P<0.05). None of the patients experienced an adverse event. CONCLUSION: Results showed that FDTV resulted in a greater reduction in resistin levels than T treatment alone.

The Actin-Binding Protein Cortactin Promotes Sepsis Severity by Supporting Excessive Neutrophil Infiltration into the Lung.

Sepsis is a systemic infection that can lead to multi-organ failure. It is characterised by an uncontrolled immune response with massive neutrophil influx into peripheral organs. Neutrophil extravasation into tissues depends on actin remodeling and actin-binding proteins such as cortactin, which is expressed ubiquitously, except for neutrophils. Endothelial cortactin is necessary for proper regulation of neutrophil transendothelial migration and recruitment to sites of infection. We therefore hypothesised that cortactin plays a crucial role in sepsis development by regulating neutrophil trafficking. Using a murine model of sepsis induced by cecal ligation and puncture (CLP), we showed that cortactin-deficient (KO) mice survive better due to reduced lung injury. Histopathological analysis of lungs from septic KO mice revealed absence of oedema, reduced vascular congestion and mucus deposition, and better-preserved alveoli compared to septic wild-type (WT) mice. Additionally, sepsis-induced cytokine storm, excessive neutrophil infiltration into the lung and oxidative stress were significantly reduced in KO mice. Neutrophil depletion 12 h after sepsis improved survival in WT mice by averting lung injury, similar to both neutrophil-depleted and non-depleted KO mice. Our findings highlight a critical role of cortactin for lung neutrophil infiltration and sepsis severity.

Annickia polycarpa extract attenuates inflammation, neutrophil recruitment, and colon damage during colitis.

Inflammatory bowel diseases (IBD) including Crohn's disease (CD) and ulcerative colitis (UC) are complex inflammatory disorders of the digestive tract. Dysfunctional intestinal epithelial barrier, uncontrolled neutrophil recruitment into the colon, and oxidative stress are major features of IBD. IBD cannot be cured, but symptoms can be alleviated with anti-inflammatory drugs, which often show adverse effects. Thus, safer alternative treatment options are needed. Given the known anti-inflammatory properties of Annickia polycarpa extract (APE), we hypothesized that APE improves the outcome of the inflammatory response during colitis. We assessed APE effects on colon histology, epithelial barrier function and neutrophil recruitment during DSS-induced colitis in mice treated with APE. APE treatment significantly reduced the disease activity index and prevented DSS-induced colon damage as evidenced by reduced colon shortening, ulcerations, crypt dysplasia, edema formation, and leukocyte infiltration. Expression of the pro-inflammatory cytokines TNF-α, IL-6, and IL-1ß were significantly diminished in APE-treated mice. Importantly, APE administration reduced neutrophil infiltration into the lamina propria leading to reduced oxidative stress, tight junction disruption and epithelial permeability in the colon. Thus, we propose APE as additional treatment strategy to attenuate colitis symptoms and enhance life quality of individuals with IBD.

HS1 deficiency protects against sepsis by attenuating neutrophil-inflicted lung damage.

Sepsis remains an important health problem worldwide due to inefficient treatments often resulting in multi-organ failure. Neutrophil recruitment is critical during sepsis. While neutrophils are required to combat invading bacteria, excessive neutrophil recruitment contributes to tissue damage due to their arsenal of molecular weapons that do not distinguish between host and pathogen. Thus, neutrophil recruitment needs to be fine-tuned to ensure bacterial killing, while avoiding neutrophil-inflicted tissue damage. We recently showed that the actin-binding protein HS1 promotes neutrophil extravasation; and hypothesized that HS1 is also a critical regulator of sepsis progression. We evaluated the role of HS1 in a model of lethal sepsis induced by cecal-ligation and puncture. We found that septic HS1-deficient mice had a better survival rate compared to WT mice due to absence of lung damage. Lungs of septic HS1-deficient mice showed less inflammation, fibrosis, and vascular congestion. Importantly, systemic CLP-induced neutrophil recruitment was attenuated in the lungs, the peritoneum and the cremaster in the absence of HS1. Lungs of HS1-deficient mice produced significantly more interleukin-10. Compared to WT neutrophils, those HS1-deficient neutrophils that reached the lungs had increased surface levels of Gr-1, ICAM-1, and L-selectin. Interestingly, HS1-deficient neutrophils had similar F-actin content and phagocytic activity, but they failed to polymerize actin and deform in response to CXCL-1 likely explaining the reduced systemic neutrophil recruitment in HS1-deficient mice. Our data show that HS1 deficiency protects against sepsis by attenuating neutrophil recruitment to amounts sufficient to combat bacterial infection, but insufficient to induce tissue damage.

ADAM17/MMP inhibition prevents neutrophilia and lung injury in a mouse model of COVID-19.

Severe coronavirus disease 2019 (COVID-19) is characterized by lung injury, cytokine storm, and increased neutrophil-to-lymphocyte ratio (NLR). Current therapies focus on reducing viral replication and inflammatory responses, but no specific treatment exists to prevent the development of severe COVID-19 in infected individuals. Angiotensin-converting enzyme-2 (ACE2) is the receptor for SARS-CoV-2, the virus causing COVID-19, but it is also critical for maintaining the correct functionality of lung epithelium and endothelium. Coronaviruses induce activation of a disintegrin and metalloprotease 17 (ADAM17) and shedding of ACE2 from the cell surface resulting in exacerbated inflammatory responses. Thus, we hypothesized that ADAM17 inhibition ameliorates COVID-19-related lung inflammation. We employed a preclinical mouse model using intratracheal instillation of a combination of polyinosinic:polycytidylic acid (poly(I:C)) and the receptor-binding domain of the SARS-CoV-2 spike protein (RBD-S) to mimic lung damage associated with COVID-19. Histologic analysis of inflamed mice confirmed the expected signs of lung injury including edema, fibrosis, vascular congestion, and leukocyte infiltration. Moreover, inflamed mice also showed an increased NLR as observed in critically ill COVID-19 patients. Administration of the ADAM17/MMP inhibitors apratastat and TMI-1 significantly improved lung histology and prevented leukocyte infiltration. Reduced leukocyte recruitment could be explained by reduced production of proinflammatory cytokines and lower levels of the endothelial adhesion molecules ICAM-1 and VCAM-1. Additionally, the NLR was significantly reduced by ADAM17/MMP inhibition. Thus, we propose inhibition of ADAM17/MMP as a novel promising treatment strategy in SARS-CoV-2-infected individuals to prevent the progression toward severe COVID-19.

Relationship between angiotensin II receptor expression and cardiovascular risk factors in Mexican patients with coronary occlusive disease.

The density of Angiotensin II (Ang) receptors on tissue surfaces is regulated by multiple hormones, cytokines and metabolic factors and is profoundly affected by various pathological conditions, such as age, diet and environmental conditions. The participation of several cardiovascular risk factors in the regulation of Angiotensin II receptor expression has been incompletely studied. We performed an ex-vivo study with human aortic postsurgical specimens to test the hypothesis that Ang AT1 and AT2 receptor expression in human aortic arteries is associated with the presence of cardiovascular risk factors. We included 31 Mexican patients with coronary artery disease. We evaluated Angiotensin II receptor expression by immunostaining and angiotensin converting enzyme insertion/deletion (ACE I/D) polymorphisms by polymerase chain reaction. AT1 and AT2 receptor expression was increased in the aortic segments from the cardiovascular patients compared with control arteries and in patients with the DD genotype. There was a correlation between increased AT1 receptor expression and the number of cardiovascular risk factors present in the patient. Furthermore, reduction of AT1 expression correlated with the number of drug combinations used in the patients. These correlations were not present with respect to AT2 receptor expression. We suggest that increased AT1 receptor expression is associated with the DD genotype. Thus the presence of several cardiovascular risk factors as well as DD genotype, induce AT1 expression increasing the probability to develop coronary occlusive disease.

Prevention of renal injury and endothelial dysfunction by chronic L-arginine and antioxidant treatment.

We evaluated the effects of vitamins with antioxidant properties (a combination of vitamins C and E) and L-arginine treatment on renal failure in mice by measuring survival rate. The molecular changes were elucidated by determining endothelial tetrahydrobiopterin (BH4) levels and nitric oxide synthase (eNOS) mRNA expression in mice with renal ablation. Previous studies have shown that endothelial dysfunction in 5/6 nephrectomized mice is associated with decreased nitric oxide (NO) bioavailability and increased vascular superoxide production. WTC57 mice were divided into three groups: Group 1 was the sham-operated group (C); Group 2 was the 5/6 nephrectomized group (Nfx); and Group 3 was a group of 5/6 nephrectomized mice, treated with L-arginine and vitamins with antioxidant properties (NfxTx; 200 mg/kg L-arginine, 83 mg/kg vitamin C, and 46.6 mg/kg vitamin E). After 20 weeks of treatment, urinary protein excretion, blood pressure, BH4 and dihydrobiopterin (BH2) levels, eNOS mRNA, oxidative stress, and survival rate were determined. An increase in urinary protein excretion, blood pressure, and oxidative stress was prevented in the NfxTx group, but not in the Nfx group. BH4 and eNOS mRNA expression was increased by 32% and 78%, respectively, in the NfxTx group. Furthermore, the treatment increased the survival rate by 33%. Our results indicate that under normal conditions, NO appears to protect renal function. However, this NO-dependent protection is lost during kidney failure, probably due to increased reactive oxygen species synthesis. The treatment restores the viability of NO and prevents the BH4 oxidation. Therefore, this treatment may represent a therapeutic approach for the management of kidney disease.

The Arp2/3 Inhibitory Protein Arpin Is Required for Intestinal Epithelial Barrier Integrity.

The intestinal epithelial barrier (IEB) depends on stable interepithelial protein complexes such as tight junctions (TJ), adherens junctions (AJ), and the actin cytoskeleton. During inflammation, the IEB is compromised due to TJ protein internalization and actin remodeling. An important actin regulator is the actin-related protein 2/3 (Arp2/3) complex, which induces actin branching. Activation of Arp2/3 by nucleation-promoting factors is required for the formation of epithelial monolayers, but little is known about the relevance of Arp2/3 inhibition and endogenous Arp2/3 inhibitory proteins for IEB regulation. We found that the recently identified Arp2/3 inhibitory protein arpin was strongly expressed in intestinal epithelial cells. Arpin expression decreased in response to tumor necrosis factor (TNF)α and interferon (IFN)γ treatment, whereas the expression of gadkin and protein interacting with protein C-kinase α-subunit 1 (PICK1), other Arp2/3 inhibitors, remained unchanged. Of note, arpin coprecipitated with the TJ proteins occludin and claudin-1 and the AJ protein E-cadherin. Arpin depletion altered the architecture of both AJ and TJ, increased actin filament content and actomyosin contractility, and significantly increased epithelial permeability, demonstrating that arpin is indeed required for maintaining IEB integrity. During experimental colitis in mice, arpin expression was also decreased. Analyzing colon tissues from ulcerative colitis patients by Western blot, we found different arpin levels with overall no significant changes. However, in acutely inflamed areas, arpin was significantly reduced compared to non-inflamed areas. Importantly, patients receiving mesalazine had significantly higher arpin levels than untreated patients. As arpin depletion (theoretically meaning more active Arp2/3) increased permeability, we wanted to know whether Arp2/3 inhibition would show the opposite. Indeed, the specific Arp2/3 inhibitor CK666 ameliorated TNFα/IFNγ-induced permeability in established Caco-2 monolayers by preventing TJ disruption. CK666 treatment also attenuated colitis development, colon tissue damage, TJ disruption, and permeability in dextran sulphate sodium (DSS)-treated mice. Our results demonstrate that loss of arpin triggers IEB dysfunction during inflammation and that low arpin levels can be considered a novel hallmark of acute inflammation.

Effect of phlorizin on SGLT2 expression in the kidney of diabetic rats.

BACKGROUND: The purpose of our study was to determine whether increased SGLT2 expression in the kidney of diabetic rats was associated with the development of hypertension and to investigate the effect of phlorizin (P) on blood pressure and SGLT2 expression in diabetic rats. METHODS: The animals were divided into two groups: Control (C) and streptozotocin-induced diabetic (D) rats were used to evaluate SGLT2 activity in brush border membrane vesicles (BBMV) using a rapid filtration technique. Others animals were divided into two groups: Normal (NSD) or high salt diet (4%)(HSD), and subdivided in four groups: C, C+P, D, D+P. Systolic blood pressure (SBP) was recorded for 30 days by the use of a telemetric system and at day 30 urine samples (24 h) were collected to evaluate renal function and SGLT2 expression in the renal cortex. RESULTS: At day 30, diabetic animals with NSD or HSD exhibited hyperglycemia, lower body weight, glycosuria, diuresis, decrease natriuresis, increased SBP values and SGLT2 expression. In diabetic rats, phlorizin treatment decreased hyperglycemia and prevented development of hypertension, decreased SGLT2 activity in BBMV but did not modify SGLT2 expression. CONCLUSIONS: In conclusion, SGLT2 inhibition prevented the development of hypertension in diabetic rats as well as hyperglycemia, suggesting a hypertensive mechanism associated with SGLT2 activity and the likelihood that increased SGLT2 expression may be associated with progression of diabetic renal complications.

Correlation between the levels of circulating adhesion molecules and atherosclerosis in hypertensive type-2 diabetic patients.

Endothelial dysfunction is a common feature in type-2 diabetic patients and in hypertension, and is associated with inflammation, increased levels of circulating soluble adhesion molecules, and atherosclerosis. The aim of this study was to evaluate the relationship between the levels of circulating soluble adhesion molecules and the degree of atherosclerosis in hypertensive type-2 diabetic patients. We studied 30 hypertensive type-2 diabetic patients in whom VCAM-1, ICAM-1, and E-selectin were measured by ELISA. Additionally, the intimal-medial thickness of both the common and internal carotid arteries was measured (B-mode ultrasound). The levels of circulating adhesion molecules and maximal carotid artery intimal-medial thicknesses were correlated using the Spearman correlation coefficient test. Statistical analysis was performed with ANOVA. We found significant correlations between ICAM-1 (r = 0.5) levels and maximal carotid artery intimal-medial thickness these patients. No correlation was observed with E-selectin and VCAM-1. Our results suggest that ICAM-1 is associated and correlated with the degree of atherosclerosis in type-2 diabetic hypertensive patients.

L-arginine and antioxidant diet supplementation partially restores nitric oxide-dependent regulation of phenylephrine renal vasoconstriction in diabetics rats.

OBJECTIVE: The increase of reactive oxygen species (ROS) in diabetes potentiates the vascular effects of phenylephrine through nitric oxide (NO) impairment, facilitating the development of diabetic nephropathy. We propose that the combination of an antioxidant and L-arginine as diet supplements could prevent the increased vascular response to phenylephrine in diabetic animals. DESIGN: Changes in the adrenergic system play an important role in the development of vascular complications in the prediabetic condition. The vasoconstrictor effects of phenylephrine are regulated by NO, and the impairment of endothelium-dependent vasodilation in diabetes is associated with ROS. SETTING: Diabetes was induced with a low dose (55 mg/kg body weight) of streptozotocin in 7-week-old rats. Diabetic rats were fed with a diet supplement containing a combination of vitamin E, vitamin C, eicosapentaenoic acid, docosahexaenoic acid, and L-arginine, and the effects on phenylephrine-induced renal vascular responses were evaluated. RESULTS: Phenylephrine increased the renal perfusion pressure of isolated perfused kidneys from diabetic rats compared with nondiabetic rats. This effect was associated with reduced nitrite release as well as reduced plasma tetrahydrobiopterin and increased superoxide anions in the renal tissue. Diet supplementation with a combination of L-arginine and vitamins in diabetic rats partially prevented the generation of superoxide associated with recovery of the renal release of NO and decreased phenylephrine-induced vasoconstrictor effects, compared with untreated diabetic rats. However, the administration of L-arginine or vitamins alone did not affect phenylephrine-induced vasoconstriction. Vitamin treatment alone did decrease superoxide generation. CONCLUSION: The protective mechanism of NO on the vasoconstrictor effects of phenylephrine in the kidney is lost during the development of diabetes, probably via the actions of ROS through a decrease in tetrahydrobiopterin, thus contributing to the pathogenesis of diabetic nephropathy. Restoration of this protective NO mechanism can be achieved by simultaneously stimulating NO synthesis and preventing the effects of ROS through the use of L-arginine and a combination of vitamins E and C as diet supplementation.