Microvascular Permeability Research Articles

Omentum-on-a-chip: A multicellular, vascularized microfluidic model of the human peritoneum for the study of ovarian cancer metastases

Epithelial ovarian cancer has the highest mortality rate of any gynecologic malignancy and most frequently metastasizes to the peritoneal cavity. Intraperitoneal metastases are highly associated with ascites, the pathologic accumulation of peritoneal fluid due to impaired drainage, increased peritoneal permeability, and tumor and stromal cytokine secretion. However, the relationship between ascites, vascular and mesothelial permeability, and ovarian cancer intraperitoneal metastases remains poorly understood. In this study, a vascularized in vitro model of the human peritoneal omentum and ovarian tumor microenvironment (TME) was employed to study stromal cell effects on tumor cell (TC) attachment and growth, as well as TC effects on vascular and mesothelial permeability in models of both early- and late-stage metastases. Control over the number of TCs seeded in the vascularized peritoneum revealed a critical cell density requirement for tumor growth, which was further enhanced by stromal adipocytes and endothelial cells found in the peritoneal omentum. This tumor growth resulted in both a physically-mediated decrease and cytokine-mediated increase in microvascular permeability, emphasizing the important and potentially opposing roles of tumor cells in ascites formation. This system provides a robust platform to elucidate TC-stromal cell interactions during intraperitoneal metastasis of ovarian cancer and presents the first in vitro vascularized model of the human peritoneum and ovarian cancer TME.

AKT/PACS2 Participates in Renal Vascular Hyperpermeability by Regulating Endothelial Fatty Acid Oxidation in Diabetic Mice.

Diabetes is a chronic metabolic disorder that can cause many microvascular and macrovascular complications, including diabetic nephropathy. Endothelial cells exhibit phenotypic and metabolic diversity and are affected by metabolic disorders. Whether changes in endothelial cell metabolism affect vascular endothelial function in diabetic nephropathy remains unclear. In diabetic mice, increased renal microvascular permeability and fibrosis, as well as increased MAMs and PACS2 in renal endothelial cells, were observed. Mice lacking PACS2 improved vascular leakage and glomerulosclerosis under high fat diet. In vitro, PACS2 expression, VE-cadherin internalization, fibronectin production, and Smad-2 phosphorylation increased in HUVECs treated with high glucose and palmitic acid (HGHF). Pharmacological inhibition of AKT significantly reduced HGHF-induced upregulation of PACS2 and p-Smad2 expression. Blocking fatty acid β-oxidation (FAO) ameliorated the impaired barrier function mediated by HGHF. Further studies observed that HGHF induced decreased FAO, CPT1α expression, ATP production, and NADPH/NADP+ ratio in endothelial cells. However, these changes in fatty acid metabolism were rescued by silencing PACS2. In conclusion, PACS2 participates in renal vascular hyperpermeability and glomerulosclerosis by regulating the FAO of diabetic mice. Targeting PACS2 is potential new strategy for the treatment of diabetic nephropathy.

Preferential uptake of SARS-CoV-2 by pericytes potentiates vascular damage and permeability in an organoid model of the microvasculature.

AimsThrombotic complications and vasculopathy have been extensively associated with severe COVID-19 infection, however the mechanisms inducing endotheliitis and the disruption of endothelial integrity in the microcirculation are poorly understood. We hypothesized that within the vessel wall, pericytes preferentially take up viral particles and mediate the subsequent loss of vascular integrity.Methods and ResultsImmunofluorescence of post-mortem patient sections were used to assess pathophysiological aspects of COVID19 infection. The effects of COVID-19 on the microvasculature were assessed using a vascular organoid model exposed to live viral particles or recombinant viral antigens. We find increased expression of the viral entry receptor ACE2 on pericytes when compared to vascular endothelium, and a reduction in the expression of the junctional protein CD144, as well as increased cell death, upon treatment with both live virus and/or viral antigens. We observe a dysregulation of genes implicated in vascular permeability including NOTCH3, angiopoietin-2 and TEK. Activation of vascular organoids with IL-1β did not have an additive effect on vascular permeability. Spike antigen was detected in some patients’ lung pericytes, which was associated with a decrease in CD144 expression and increased platelet recruitment and VWF deposition in the capillaries of these patients, with thrombi in large vessels rich in VWF and fibrin.ConclusionsTogether our data indicates that direct viral exposure to the microvasculature modelled by organoid infection and viral antigen treatment result in pericyte infection, detachment, damage and cell death, disrupting pericyte-endothelial cell crosstalk and increasing microvascular endothelial permeability, which can promote thrombotic and bleeding complications in the microcirculation.Translational PerspectiveEndotheliitis is a serious complication of severe COVID-19 patients which remains poorly understood. We identify a pericyte mediated mechanism by which the vasculature becomes compromised, contributing to thrombotic complications, highlighting important avenues for the development of therapies.

Abstract 2469: Image-guided focused ultrasound-mediated drug delivery for improved cancer treatment

Abstract Introduction: The first option for cancer treatment is often chemotherapy. However, studies have shown that only 1% of the injected dose reaches the target cancer as the tumor microenvironment presents a physical barrier for optimal drug delivery. Focused ultrasound (FUS) in combination with microbubble (MB) contrast agents is an emerging therapy to improve drug delivery by temporarily increasing microvascular permeability. This research details the development and testing of a novel ultrasound (US) image-guided FUS system and method for enhancing drug delivery to tumor tissue and volume space with comparison to a 2-dimensional (2-D) US therapeutic technology. Methods: Real-time US therapy was implemented on a programmable US system (Vantage 256, Verasonics Inc) equipped with a dual US transducer configuration for interleaved anatomical imaging and volumetric treatment delivery (HIFU-Plex, Sonic Concepts Inc). Both US imaging and therapeutic transducers are co-registered 128 element arrays with center frequencies of 3.5 and 2.0 MHz, respectively. The latter is a concentric array that enables beam steering in 3-dimensional (3-D) space. US treatment was performed at a peak negative pressure of 0.7 MPa (mechanical index, MI of 0.45), pulse repetition frequency of 10 Hz, and duty cycle of 10%. BALB/c mice (N = 22, Charles River Laboratory) were implanted with 2.0 × 105 breast cancer cells (4T1, ATCC). Once tumors reached 0.6 cm in size, mice were randomly divided into a 3-D or 2-D US therapy group or sham control. US therapy was performed following an intravascular injection of microbubbles (Definity, Lantheus Medical Imaging) and IR-780 dye. Note microbubbles function as a therapeutic mediator whereas the fluorescent dye represents a surrogate small molecule drug. Live animal fluorescent imaging was performed at baseline before US therapy (0 h) and again at 1, 24, and 48 h. Following the 48 h timepoint, animals were euthanized and tumors surgically excised for ex vivo analysis. Results: 3-D US-mediated therapy improved molecular delivery to tumor tissue by 150 and 180% at 24 and 48 h, respectively, when compared to our previously established 2-D US therapeutic approach (p = 0.22) or sham therapy (p = 0.07). A similar trend was observed during ex vivo imaging of excised tumor samples treated with 3-D US therapy as compared to the 2-D US therapeutic approach (p = 0.41) or sham therapy (p = 0.04). Dye extraction further confirmed these observations (p > 0.12). Conclusions: 3-D US therapy improved molecular delivery to the tumor volume compared to our previously established 2-D US-based method as confirmed by a series of optical imaging studies. Citation Format: Ryan Margolis, Junjie Li, Lokesh Basavarajappa, Kenneth Hoyt. Image-guided focused ultrasound-mediated drug delivery for improved cancer treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2469.

Stimulatory G-Protein α Subunit Modulates Endothelial Cell Permeability Through Regulation of Plasmalemma Vesicle-Associated Protein.

Endothelial cell leakage occurs in several diseases. Intracellular junctions and transcellular fashion are involved. The definite regulatory mechanism is complicated and not fully elucidated. The alpha subunit of the heterotrimeric G-stimulatory protein (Gsα) mediates receptor-stimulated production of cyclic adenosine monophosphate (cAMP). However, the role of Gsα in the endothelial barrier remains unclear. In this study, mice with knockout of endothelial-specific Gsα (GsαECKO) were generated by crossbreeding Gsαflox/flox mice with Cdh5-CreERT2 transgenic mice, induced in adult mice by tamoxifen treatment. GsαECKO mice displayed phenotypes of edema, anemia, hypoproteinemia and hyperlipoproteinemia, which indicates impaired microvascular permeability. Mechanistically, Gsα deficiency reduces the level of endothelial plasmalemma vesicle-associated protein (PLVAP). In addition, overexpression of Gsα increased phosphorylation of cAMP response element-binding protein (CREB) as well as the mRNA and protein levels of PLVAP. CREB could bind to the CRE site of PLVAP promoter and regulate its expression. Thus, Gsα might regulate endothelial permeability via cAMP/CREB-mediated PLVAP expression.

Protective effect of Ulinastatin on acute lung injury in diabetic sepsis rats

This study explored the protective effect and its possible mechanism of ulinastatin (UTI) on acute lung injury (ALI) in type 2 diabetes mellitus (DM) sepsis rats. Following treatment with UTI, the wet/dry weight (W/D) ratio, pathological changes, hypoxia-inducible factor-1ɑlpha (HIF-1ɑ) protein and Toll-like receptor 4 (TLR4) mRNA expression of lung tissues, the expression levels of interleukin-1beta (IL-1ß), IL-18, and tumor necrosis factor-alpha (TNF-ɑ), the contents of malondialdehyde (MDA) and superoxide dismutase (SOD) in serum were detected in type 2 DM sepsis rats. It was found that rats with type 2 DM and sepsis showed obvious damage in lung tissues with significantly increased inflammatory cells, necrosis, and swelling of alveolar epithelial cells, but UTI decreased the lung damage induced by DM and sepsis. In addition, compared with the control, the W/D ratio, serum IL-1ß, IL-18 and TNF-ɑ contents, HIF-1ɑ protein expression, TLR4 mRNA expression, pulmonary microvascular permeability, MDA content in serum in type 2 DM and sepsis groups were significantly increased in type 2 DM sepsis rats (p < 0.05). However, compared with the groups with type 2 DM sepsis, the W/D ratio, serum IL-1ß, IL-18, TNF-ɑ contents, HIF-1ɑ protein expression, TLR4 mRNA expression, and pulmonary microvascular permeability in UTI-treated group were significantly decreased, but the activity of SOD increased (p < 0.05). This study indicates that UTI can effectively reduce ALI induced by diabetic sepsis in rats through inhibiting inflammatory response, reducing oxidative stress, regulating hypoxia response pathway, and improving pulmonary microvascular permeability.

MiR-18a-5p Reduces Inflammatory Response and Human Dermal Microvascular Endothelial Cells Permeability by Targeting Thrombospondin 1 in Chronic Idiopathic Urticaria

Chronic idiopathic urticaria (CIU) is a skin disease that has an acute attack when patient encounters an allergen. Previous research demonstrated that microRNA (miRNA) has a crucial role in CIU occurrence and development. This study aimed to investigate the role and mechanism of miR-18a-5p in CIU. StarBase and dual luciferase reporter gene assay confirmed that thrombospondin 1 (THBS1) was a direct target of miR-18a-5p. miR-18a-5p negatively regulated THBS1 expression in human mast cell line HMC-1 and human dermal microvascular endothelial cells (HDMECs). Further analysis indicated that miR-18a-5p mimic significantly reduced inflammatory factors including interleukin (IL)-6, IL-8, IL-1β and tumor necrosis factor (TNF)-α release, decreased the degranulation rate and histamine release rate of HMC-1 cells. Besides, we found that miR-18a-5p reduced the permeability of HDMECs by suppressing THBS1 expression. Finally, the data indicated that miR-18a-5p inhibited the transforming growth factor beta (TGF-β)/SMAD family member 3 (Smad3) signaling pathway in HDMECs by inhibiting the expression of THBS1. Taken together, the results suggested that miR-18a-5p reduced inflammation and human skin microvascular endothelial cell permeability in CIU by targeting THBS1. miR-18a-5p might be a new target for CIU treatment.

Blocking P2X7 receptor with AZ 10606120 exacerbates vascular hyperpermeability and inflammation in murine polymicrobial sepsis.

Sepsis is a devastating disease with high morbidity and mortality and no specific treatments. The pathophysiology of sepsis involves a hyperinflammatory response and release of damage‐associated molecular patterns (DAMPs), including adenosine triphosphate (ATP), from activated and dying cells. Purinergic receptors activated by ATP have gained attention for their roles in sepsis, which can be pro‐ or anti‐inflammatory depending on the context. Current data regarding the role of ATP‐specific purinergic receptor P2X7 (P2X7R) in vascular function and inflammation during sepsis are conflicting, and its role on the endothelium has not been well characterized. In this study, we hypothesized that the P2X7R antagonist AZ 10606120 (AZ106) would prevent endothelial dysfunction during sepsis. As proof of concept, we first demonstrated the ability of AZ106 (10 µM) to prevent endothelial dysfunction in intact rat aorta in response to IL‐1β, an inflammatory mediator upregulated during sepsis. Likewise, blocking P2X7R with AZ106 (10 µg/g) reduced the impairment of endothelial‐dependent relaxation in mice subjected to intraperitoneal injection of cecal slurry (CS), a model of polymicrobial sepsis. However, contrary to our hypothesis, AZ106 did not improve microvascular permeability or injury, lung apoptosis, or illness severity in mice subjected to CS. Instead, AZ106 elevated spleen bacterial burden and circulating inflammatory markers. In conclusion, antagonism of P2X7R signaling during sepsis appears to disrupt the balance between its roles in inflammatory, antimicrobial, and vascular function.

Clinicopathological alteration of symptoms with serotype among dengue infected pediatric patients

Dengue fever is a self-limiting, acute febrile illness caused by an arbovirus. This infection may be asymptomatic or symptomatic with its potential life-threatening form as DHF/DSS. Severe dengue cases occur typically in children due to overproduction of proinflammatory and anti-inflammatory cytokines (called cytokines storm) as well as increased microvascular permeability in them. This study aimed to find circulating dengue serotype and their clinicopathological association among pediatric patients admitted to tertiary care hospitals in Kolkata, India. Overall, 210 patients were approached, among them, 170 dengue suspected children admitted to three tertiary care hospitals were included in this study. Dengue samples were screened for the presence of dengue NS1 antigen and IgM antibodies by enzyme-linked immunosorbent assay. Viral RNA was extracted from NS1 seropositive serum samples and subjected to molecular serotyping by semi-nested reverse-transcription polymerase chain reaction. All patients were followed up for clinical manifestations and biochemical parameters associated with dengue. Cocirculation of all four serotypes was observed and DENV2 was the major circulating strain. Physiological classification of associated clinical symptoms was done as per WHO guideline and represented as a percentage variable. A multivariate logistic regression approach was used for making a regression model including dengue-associated clinical symptoms with dengue positivity or negativity as dependent variables. Thrombocytopenia was observed in 69% of patients and the commonest bleeding manifestation was petechia. Liver function profiles of infected patients were observed during follow-up and represented using a box plot. A significant change in trends of dengue-associated clinical manifestations and differential expression of liver functional profile with different phases of transition of dengue fever was observed in this study population.

URB597 and Andrographolide Improve Brain Microvascular Endothelial Cell Permeability and Apoptosis by Reducing Oxidative Stress and Inflammation Associated with Activation of Nrf2 Signaling in Oxygen-Glucose Deprivation.

Ischemic stroke, a cerebrovascular disease worldwide, triggers a cascade of pathophysiological events, including blood-brain barrier (BBB) breakdown. Brain microvascular endothelial cells (BMECs) play a vital role in maintaining BBB function. The injury of BMECs may worsen neurovascular dysfunction and patients' prognosis. Therefore, uncover the principal molecular mechanisms involved in BBB disruption in stroke becomes pressing. The endocannabinoid system (ECS) has been implicated in increasingly physiological functions, both in neurometabolism and cerebrovascular regulation. Modulating its activities by the fatty acid amide hydrolase (FAAH) shows anti-inflammatory characteristics. Andrographolide (AG), one Chinese herbal ingredient, has also attracted attention for its role in immunomodulatory and as a therapeutic target in BBB disorders. Recently, the FAAH inhibitor URB597 and AG have important regulatory effects on neuronal and vascular cells in ischemia. However, the effects of URB597 and AG on BMEC permeability and apoptosis in oxygen-glucose deprivation (OGD) and the underlying mechanisms remain unclear. To address these issues, cultured BMECs (bEnd.3 cells) were exposed to OGD. The cell viability, permeability, tube formation, and apoptosis were assessed following treatment with URB597, AG, and cotreatment. Mitochondrial membrane potential (MMP), reactive oxygen species (ROS), superoxide dismutase (SOD), catalase (CAT), malondialdehyde (MDA), proinflammatory factors, tight junction (TJ) proteins, and oxidative stress-mediated Nrf2 signaling were also investigated. Results revealed that OGD broke the endothelial barrier, cell viability, MMP, and tube formation, which was reversed by URB597 and AG. OGD-induced enhancement of ROS, MDA, and apoptosis was reduced after drug interventions. URB597 and AG exhibited antioxidant/anti-inflammatory and mitochondrial protective effects by activating Nrf2 signaling. These findings indicated that URB597 and AG protect BMECs against OGD-induced endothelial permeability impairment and apoptosis by reducing mitochondrial oxidative stress and inflammation associated with activation of Nrf2 signaling. URB597 and AG showing the vascular protection may have therapeutic potential for the BBB damage in ischemic cerebrovascular diseases.

Troponin T Is Negatively Associated With 3 Tesla Magnetic Resonance Peripheral Nerve Perfusion in Type 2 Diabetes

ObjectiveThe pathogenesis of diabetic polyneuropathy (DN) is poorly understood and given the increasing prevalence of DN, there is a need for clinical or imaging biomarkers that quantify structural and functional nerve damage. While clinical studies have found evidence of an association between elevated levels of troponin T (hsTNT) and N-terminal pro brain natriuretic peptide (proBNP) with microvascular compromise in type 2 diabetes (T2D), their implication in mirroring DN nerve perfusion changes remains unclear. The objective of this study was, therefore, to investigate whether hsTNT and proBNP assays are associated with MRI nerve perfusion in T2D.MethodsIn this prospective cross-sectional single-center case-control study, 56 participants (44 with T2D, 12 healthy control subjects) consented to undergo magnetic resonance neurography (MRN) including dynamic contrast-enhanced (DCE) perfusion imaging of the right leg. Using the extended Tofts model, primary outcome parameters that were quantified are the sciatic nerve’s microvascular permeability (Ktrans), the extravascular extracellular volume fraction (ve), and the plasma volume fraction (vp), as well as hsTNT and proBNP values from serological workup. Further secondary outcomes were clinical, serological, and electrophysiological findings.ResultsIn T2D patients, hsTNT was negatively correlated with Ktrans (r=-0.38; p=0.012) and ve (r=-0.30; p=0.048) but not with vp (r=-0.16; p=0.294). HsTNT, Ktrans, and ve were correlated with peroneal nerve conduction velocities (NCVs; r=-0.44; p=0.006, r=0.42; p=0.008, r=0.39; p=0.014), and tibial NCVs (r=-0.38;p=0.022, r=0.33; p=0.048, r=0.37; p=0.025). No such correlations were found for proBNP.ConclusionsThis study is the first to find that hsTNT is correlated with a decrease of microvascular permeability and a reduced extravascular extracellular volume fraction of nerves in patients with T2D. The results indicate that hsTNT may serve as a potential marker for the assessment of nerve perfusion in future studies on DN.

Mechanism of Fructus Mume Pills Underlying Their Protective Effects in Rats with Acetic Acid-Inducedulcerative Colitis via the Regulation of Inflammatory Cytokines and the VEGF-PI3K/Akt-eNOS Signaling Pathway.

Background Fructus mume pills (FMPs) have been clinically proven to be effective for treating ulcerative colitis (UC). However, the therapeutic and protective mechanisms have not been fully studied. Aim We aimed to explore the mechanism of FMPs in an acetic acid (AA)-induced ulcerative colitis rat model. Methods The targets, GO terms, and KEGG pathways for the FMPs and UC were screened and constructed using network pharmacology. A possible mechanism was verified in a 4% AA-induced colitis rat model. Colitis activity and state were evaluated using the disease activity index, and colon ulceration and intestinal mucosal damage were determined by histopathological observation through HE, AB-PAS, and Masson pathological staining. The concentrations of TNF-α, IL-6, IL-8, IL-10, MPO, MMP9, CXCR1, eNOS, and VEGF were measured to evaluate vascular permeability effects. Results The network pharmacology results showed 108 active compounds, and 139 FMP-related targets were identified. Twenty-nine targets were identified for FMPs against UC, which included MMP9, MMP3, ESR1, PTGS1, PPARA, MPO, and NOS2. A total of 1,536 GO terms and 41 pathways were associated with FMP treatment of UC. The pharmacological evaluation showed that FMPs attenuated inflammation in AA-induced colitis by reducing the serum concentrations of TNF-α, IL-6, IL-8, and IL-10 and the colonic concentrations of MPO, MMP9, and CXCR1. FMPs ameliorated hyperpermeability by reducing the colonic VEGF and eNOS concentrations. FMPs also significantly decreased the VEGFA, VEGFR2, Src, and eNOS protein expressions in colon tissue through the VEGF-PI3K/Akt-eNOS signaling pathway. Conclusion These results suggest that FMPs control UC inflammation by regulating inflammatory cytokine concentrations. FMPs alleviate AA-induced UC by regulating microvascular permeability through the VEGF-PI3K/Akt-eNOS signaling pathway.

ENOS Location is a Fundamental Mechanism for Inactivation of Inflammation‐induced Hyperpermeability

Inflammation disrupts the endothelial barrier and increases microvascular permeability (hyperpermeability), leading to tissue edema. Mechanisms for the onset of hyperpermeability have been the focus of many studies. However, the major pathological consequences are related to impairment in terminating hyperpermeability, rather than its onset, and to sustained inflammation. Therefore, we are innovatively studying mechanisms involved in the inactivation of hyperpermeability and restoration of endothelial barrier and normal microvascular permeability. We demonstrated that agonist‐induced translocation of eNOS (endothelial nitric oxide synthase) from cell membrane to cytosol initiates hyperpermeability. We hypothesize that agonist signaling for hyperpermeability also initiates a delayed increase in [cAMP], causing translocation of eNOS and Epac1 (exchange protein activated by cAMP) from cytosol to the cell membrane, which inactivates hyperpermeability. We tested whether platelet‐activating factor (PAF) induced hyperpermeability first and in delayed manner increased cAMP concentration to selectively stimulate Epac1 leading to translocation of eNOS from cytosol back to the cell membrane. In addition, we studied whether vasodilator‐stimulated phosphoprotein (VASP) is involved in the inactivation of hyperpermeability since PAF‐induced hyperpermeability in VASP‐KO endothelial cells remains elevated after Epac1 stimulation. We used human microvascular endothelial cells (HMVEC) and ECV‐304 cells transfected with green fluorescent protein (GFP)‐conjugated eNOS or the constructs GFPeNOSG2A (which anchors eNOS to the cytoplasm, ECV‐GFPeNOS‐G2A) and GFPeNOS‐CAAX (which anchors eNOS to the plasma membrane, ECV‐GFPeNOS‐CAAX) to study the restoration of endothelial barrier after hyperpermeability induced by PAF or vascular endothelial growth factor (VEGF). The time‐correlation between [NO] and [cAMP] in PAF or VEGF‐stimulated HMVEC and ECV‐eNOSGFP indicates that NO increase precedes [cAMP] increase. The increase in [cAMP] in HMVEC and ECV‐eNOSGFP correlates with a close proximity between eNOS, Epac1 and VASP, and both eNOS and Epac1 translocate to the cell membrane. None of these observations occur when eNOS is anchored to the cell membrane or cytosol. Furthermore, stimulation of Epac1 in HMVEC and ECV‐eNOSGFP inactivates their hyperpermeability response to PAF or VEGF. Stimulation of Epac1 in ECV‐GFPeNOS‐CAAX, after PAF application, inhibits hyperpermeability, while Epac1 stimulation does not reduce PAF‐elicited hyperpermeability in ECV‐GFPeNOS‐G2A. Stimulation of Epac1, after application of PAF, in the in vivo male and female hamster cheek pouch prevents hyperpermeability. In conclusion, our results support the concept that eNOS, cAMP, Epac1 and VASP work in synchrony to terminate hyperpermeability by translocating eNOS back to the cell membrane, and the overarching concept that location of eNOS is an important determinant in the regulation of microvascular permeability.

Pharmacological evaluation of Thuja occidentalis for the attenuation of nephropathy in streptozotocin-induced diabetes rats

In diabetic nephropathy (DN), chronic hyperglycemia causes oxidative stress and dyslipidemia, resulting in chronic renal damage. To mitigate the effects of hyperglycemia-induced kidney impairment, it may be a potential mechanism for future research This study aims to evaluate the nephroprotective efficacy of Thuja occidentalis in streptozotocin (STZ)-nicotinamide (NAD)-induced DN in male Wistar rats. Single dose of STZ (65 mg/kg, i.p.) was used to induce DN in Wistar rats. Serum insulin, glucose, renal function test, lipid profile, oxidative stress, inflammatory markers & histopathology were used to evaluate nephropathy. Wistar rats were treated with varying doses of hydroalcoholic extracts of Thuja occidentalis (HAETO) and Glimepiride for 30 days. Thuja occidentalis considerably correct the lipid profile levels and oxidative stress caused by hyperglycemia and the restoration of renal functions, indicating that it effectively treats DN. These findings indicate that the HAETO effectively inhibits the development of DN. The hypoglycemic, antioxidant, and anti-inflammatory properties of Thuja occidentalis are thought to be responsible for the nephroprotective benefit. • Accumulation of AGE's plays a significant role in establishing Diabetes Nephropathy. • Oxidative stress may be a promising strategy for reversing hyperglycemia-induced kidney impairment. • STZ is used in experimental animals to induce diabetes mellitus. • In diabetes, enhanced levels of TNF-α and interleukins cause microvascular permeability and renal function loss. • Thuja Occidentalis's antioxidant activity may play a crucial part in the recovery of kidney tissues.

Skeletal Muscle Injury is Sufficient for Angiogenesis but Myofiber Regeneration is Required for Restoring Microvascular Integrity

Acute injury of skeletal muscle damages myofibers, microvessels, and supporting tissue components. To restore the structural and functional integrity of intact muscle, each tissue component must regenerate. In adult skeletal muscle, resident muscle stem cells (satellite cells) efficiently regenerate myofibers within 10 days post injury (dpi) and microvascular integrity recovers by 21 dpi. However, the requirement for crosstalk between regenerating myofibers and microvessels is poorly understood. We tested the hypothesis that myofiber regeneration is necessary for revascularization and restoration of microvascular integrity. To ablate satellite cells and impair myofiber regeneration, we studied male and female mice (age, ~4 mo) in which tamoxifen‐dependent Cre recombination induced the expression of diphtheria toxin in quiescent satellite cells, killing them prior to injury. For acute muscle injury, the gluteus maximus muscle (GM) was injected with BaCl2 (1.2%, 75 µL). Microvascular perfusion and permeability were evaluated by injecting fluorescent dextrans (70 &amp; 155 kDa) into the circulation during intravital imaging of the GM at defined timepoints following injury. Despite the absence of myofiber regeneration, sprouting angiogenesis was robust and capillary perfusion was restored within 7 dpi, as observed in wild‐type mice. However, the permeability barrier of nascent microvessels was compromised as shown by profuse leakage of both fluorescent dextrans. At 21 dpi, the absence of regenerated myofibers was accompanied by sustained inflammation and myosteatosis. Areas of ectopic adipocyte accumulation were hypervascular and localized around venules. Adjacent areas contained empty myofiber “sheaths” which were underperfused; residual microvascular networks exhibited pruning. These findings suggest that, following acute injury of adult skeletal muscle, revascularization occurs in the absence of myogenesis but is defective and nascent vessels are unstable. We conclude that myofiber regeneration is essential to recovering a functionally intact microcirculation following muscle injury.

SMAD3 is a Mediator of TGF‐β ‐induced Barrier Dysfunction and Hyperpermeability in Microvascular Endothelial Cells

The endothelial transcription factor ETS (Erythroblast‐transformation specific)‐related gene (ERG) regulates canonical TGFβ‐SMAD signaling, by repressing SMAD3 activity. SMAD proteins function as important mediators of intracellular signal transduction of TGF‐β. Following their phosphorylation by TGF‐β receptor‐I, Receptor‐regulated SMADs (including SMAD2 and SMAD3 proteins) form a heteromeric complex with co‐SMAD and then translocate into the nucleus where they bind and regulate the expression of multiple target genes. Recent studies from our lab have demonstrated that ERG is a critical regulator of permeability in human lung microvascular endothelial cells (HLMECs) where ERG gene activation provided protection to the barrier. We have hypothesized that the protective effects of ERG on barrier integrity may be mediated via inhibiting TGF‐β/SMAD3 signaling pathway. To study the role of SMAD3 in microvascular endothelial barrier functions and permeability, HLMECs were grown on Transwell inserts and other cell culture platforms. Monolayer permeability was evaluated based on FITC‐albumin extravasation and measurement of its fluorescence intensity. TGF‐β (1‐50 ng/ml) induced a dose‐dependent increase in monolayer hyperpermeability. TGF‐β‐induced hyperpermeability was attenuated by a SMAD3 pharmacological inhibitor (SIS3) or CRISPR/cas9‐mediated knockdown of SMAD3. Immunoblot analysis demonstrated highly efficient SMAD3 knockdown by SMAD3 CRISPR/Cas9. TGF‐β treatment resulted in significant increase in matrixmetalloproteinase‐9 (MMP‐9) activity and the effect was decreased by SMAD3 inhibition. MMP‐9 is known to proteolytically breakdown the adherens junction proteins in microvascular endothelial cells, resulting in hyperpermeability. These results demonstrate that SMAD3 is a mediator of TGF‐β signaling‐induced loss of endothelial barrier integrity and permeability in microvascular endothelial cells. It also suggests that one of the downstream effects of TGFβ‐SMAD signaling is the activation of MMP‐9 which mediates loss of adherens junctional integrity.

Loss of Endothelial CFTR Drives Barrier Failure and Edema Formation in Lung Infection and Can Be Targeted by CFTR Potentiation

Pneumonia is the most common cause of the acute respiratory distress syndrome (ARDS), a potentially fatal lung disease characterized by hyperinflammation and endothelial barrier failure. Infectious and inflammatory stimuli can cause rapid downregulation of cystic fibrosis transmembrane conductance regulator (CFTR), and inhibition of CFTR has been proposed to increase lung microvascular endothelial permeability in vitro. Here, we identify loss of lung microvascular endothelial CFTR as important pathomechanism in lung barrier failure in pneumonia‐induced ARDS, delineate the molecular signaling pathway underlying this effect and identify CFTR potentiation as novel therapeutic strategy in ARDS.CFTR was downregulated in endothelial cells following Streptococcus pneumoniae(S.pn.) infection in human and murine lung tissue. Isolated perfused lungs revealed that CFTR inhibition increased endothelial permeability in parallel with intracellular Cl‐ and Ca2+concentrations ([Cl‐]i, [Ca2+]i). Inhibition of the Cl‐ sensitive with‐no‐lysine kinase 1 (WNK1) replicated the effect of CFTR inhibition on endothelial permeability and endothelial [Ca2+]i while WNK1 activation attenuated it. Endothelial [Ca2+]i transients and permeability in response to inhibition of either CFTR or WNK1 were prevented by inhibition of the cation channel transient receptor potential vanilloid 4 (TRPV4). Mice deficient in Trpv4 (Trpv4‐/‐) developed less lung edema and protein leak than their wild‐type littermates following infection with S. pn. Conversely, lungs of heterozygous Wnk1‐deficient mice (Wnk1+/‑) showed spontaneous leak. The CFTR potentiator ivacaftor prevented CFTR loss and reduced endothelial leak in response to pneumolysin (PLY), a key S.pn. virulence factor, or plasma from COVID‐19 patients in vitro, and prevented lung edema and protein leak after S. pn.infection in vivo.Lung infection causes rapid loss of CFTR that promotes lung edema formation through intracellular Cl‐accumulation, inhibition of WNK1 and subsequent disinhibition of TRPV4, resulting in endothelial Ca2+ influx and vascular barrier failure. Ivacaftor prevents CFTR loss and may thus present a promising therapeutic strategy in ARDS due to severe pneumonia including COVID‐19.

In Vitro Screening Identifies TRPV4 and PAR1 as Targets for Endothelial Barrier Stabilization in COVID‐19

Study objectiveEndothelial dysfunction and increased microvascular permeability are hallmarks of severe COVID‐19. At present, the underlying mechanisms of endothelial barrier failure in COVID‑19 remain elusive. Here, we show that increased thrombin activity in plasma from severe COVID‐19 patients activates endothelial protease‐activated receptor (PAR1), which mediates barrier failure by triggering TRPV4‐mediated Ca2+ influx in lung microvascular endothelial cells.MethodsCitrate plasma was sampled as part of the Pa‐COVID‐19 cohort study (ethics approval EA2/066/20) from patients with severe COVID‐19 (high flow O2 or mechanically ventilated; WHO severity score: 5‐7) COVID‑19. Plasma samples were diluted to 10% (v/v) in cell culture medium without FCS and tested for their ability to disrupt barrier integrity of primary human pulmonary microvascular endothelial cells (HPMEC) monolayers by electrical cell‐substrate impedance sensing (ECIS), immunofluorescence for endothelial VE‐cadherin and F‐actin, western blot analyses of PAR‐1 cleavage, and real‐time Ca2+ imaging. Plasma from healthy donors served as control.ResultsCOVID‐19 plasma had elevated thrombin activity while levels of antithrombin III, a key anti‐coagulant with thromboprotective function were decreased. COVID‐19 plasma caused endothelial barrier dysfunction as measured by ECIS and gap formation in HPMEC monolayers. Endothelial barrier disruption and endothelial Ca2+ influx in response to COVID‐19 plasma could be blocked by selective antagonists targeting thrombin (Argatroban), its receptor PAR1 (SCH79797), or TRPV4 (HC‐067047).ConclusionHere, we identify a novel signaling axis involving thrombin, its receptor PAR1, and TRPV4 as mechanism for increased microvascular permeability in COVID‑19. Targeting this signaling axis in endothelial barrier failure may provide a promising adjunctive therapy in COVID‐19.

Sciatic nerve microvascular permeability in type 2 diabetes decreased in patients with neuropathy.

ObjectivesClinical and histological studies have found evidence that nerve ischemia is a major contributor to diabetic neuropathy (DN) in type 2 diabetes (T2D). The aim of this study was to investigate peripheral nerve microvascular permeability using dynamic contrast enhanced (DCE) magnetic resonance neurography (MRN) to analyze potential correlations with clinical, electrophysiological, and demographic data.MethodsSixty‐five patients (35/30 with/without DN) and 10 controls matched for age and body mass index (BMI) underwent DCE MRN of the distal sciatic nerve with an axial T1‐weighted sequence. Microvascular permeability (K trans), plasma volume fraction (v p), and extravascular extracellular volume fraction (v e) were determined with the extended Tofts model, and subsequently correlated with clinical data.Results K trans and v e were lower in T2D patients with DN compared to patients without DN (0.037 min−1 ± 0.010 vs. 0.046 min−1 ± 0.014; p = 0.011, and 2.35% ± 3.87 vs. 5.11% ± 5.53; p = 0.003, respectively). In individuals with T2D, K trans correlated positively with tibial, peroneal, and sural NCVs (r = 0.42; 95%CI = 0.18 to 0.61, 0.50; 95%CI = 0.29 to 0.67, and 0.44; 95%CI = 0.19 to 0.63, respectively), with tibial and peroneal CMAPs (r = 0.27; 95%CI = 0.01 to 0.49 and r = 0.32; 95%CI = 0.07 to 0.53), and with the BMI (r = 0.47; 95%CI = 0.25 to 0.64). Negative correlations were found with the neuropathy deficit score (r = −0.40; 95%CI = −0.60 to −0.16) and age (r = −0.51; 95%CI = −0.67 to −0.31). No such correlations were found for v p.ConclusionThis study is the first to find associations of MR nerve perfusion parameters with clinical and electrophysiological parameters related to DN in T2D. The results indicate that a decrease in microvascular permeability but not plasma volume may result in nerve ischemia that subsequently causes demyelination.

DOCK4 Regulation of Rho GTPases Mediates Pulmonary Vascular Barrier Function.

The vascular endothelium maintains tissue-fluid homeostasis by controlling the passage of large molecules and fluid between the blood and interstitial space. The interaction of catenins and the actin cytoskeleton with VE-cadherin (vascular endothelial cadherin) is the primary mechanism for stabilizing AJs (adherens junctions), thereby preventing lung vascular barrier disruption. Members of the Rho (Ras homology) family of GTPases and conventional GEFs (guanine exchange factors) of these GTPases have been demonstrated to play important roles in regulating endothelial permeability. Here, we evaluated the role of DOCK4 (dedicator of cytokinesis 4)-an unconventional Rho family GTPase GEF in vascular function. We generated mice deficient in DOCK4' used DOCK4 silencing and reconstitution approaches in human pulmonary artery endothelial cells' used assays to evaluate protein localization, endothelial cell permeability, and small GTPase activation. Our data show that DOCK4-deficient mice are viable. However, these mice have hemorrhage selectively in the lung, incomplete smooth muscle cell coverage in pulmonary vessels, increased basal microvascular permeability, and impaired response to S1P (sphingosine-1-phosphate)-induced reversal of thrombin-induced permeability. Consistent with this, DOCK4 rapidly translocates to the cell periphery and associates with the detergent-insoluble fraction following S1P treatment, and its absence prevents S1P-induced Rac-1 activation and enhancement of barrier function. Moreover, DOCK4-silenced pulmonary artery endothelial cells exhibit enhanced basal permeability in vitro that is associated with enhanced Rho GTPase activation. Our findings indicate that DOCK4 maintains AJs necessary for lung vascular barrier function by establishing the normal balance between RhoA (Ras homolog family member A) and Rac-1-mediated actin cytoskeleton remodeling, a previously unappreciated function for the atypical GEF family of molecules. Our studies also identify S1P as a potential upstream regulator of DOCK4 activity.