Endothelial Membrane Research Articles

Mycolactone causes destructive Sec61-dependent loss of the endothelial glycocalyx and vessel basement membrane: a new indirect mechanism driving tissue necrosis in Mycobacterium ulcerans infection.

The drivers of tissue necrosis in Mycobacterium ulcerans infection (Buruli ulcer disease) have historically been ascribed solely to the directly cytotoxic action of the diffusible exotoxin, mycolactone. However, its role in the clinically-evident vascular component of disease aetiology remains poorly explained. We have now dissected mycolactone's effects on primary vascular endothelial cells in vitro and in vivo. We show that mycolactone-induced changes in endothelial morphology, adhesion, migration, and permeability are dependent on its action at the Sec61 translocon. Unbiased quantitative proteomics identified a profound effect on proteoglycans, driven by rapid loss of type II transmembrane proteins of the Golgi, including enzymes required for glycosaminoglycan (GAG) synthesis, combined with a reduction in the core proteins themselves. Loss of the glycocalyx is likely to be of particular mechanistic importance, since knockdown of galactosyltransferase II (beta-1,3-galactotransferase 6; B3GALT6), the GAG linker-building enzyme, phenocopied the permeability and phenotypic changes induced by mycolactone. Additionally, mycolactone depleted many secreted basement membrane components and microvascular basement membranes were disrupted in vivo. Remarkably, exogenous addition of laminin-511 reduced endothelial cell rounding, restored cell attachment and reversed the defective migration caused by mycolactone. Hence supplementing mycolactone-depleted extracellular matrix may be a future therapeutic avenue, to improve wound healing rates.

Repeated treatment with VEGF receptor inhibitors induces phenotypic changes in endothelial cells and pericytes in the rat retina

Abnormal ocular angiogenesis is a major cause of visual impairment and vision loss in neovascularization-related diseases. Currently, anti-vascular endothelial growth factor (VEGF) drugs are used to treat ocular neovascularization, but repeated injections are needed to maintain their therapeutic effects. However, repeated injection of anti-VEGF drugs may affect the retinal blood vessel phenotype and diminish therapeutic effects. In this study, we aimed to investigate the phenotypic changes in endothelial cells and pericytes caused by the repeated interruption of the VEGF receptor signaling pathway in neonatal rats. KRN633 (10 mg/kg), a VEGF receptor tyrosine kinase inhibitor, was subcutaneously administered on postnatal day (P)-7 and P8 (first round), P14 and P15 (second round), and P21 and P22 (third round). The rat eyes were collected on P7, P9, P14, P16, P21, P23, P28, and P35. Using retinal flat-mount specimens stained with specific markers for vascular endothelial cells, basement membranes, and pericytes, the arteriolar tortuosity, capillary area density, and distribution of pericytes were evaluated. Significant loss of capillaries was observed the day after the first round of KRN633 treatment, after which aggressive angiogenesis occurred, leading to the formation of tortuous arterioles. Rats that completed second and third rounds of KRN633 treatment showed more severe abnormalities in the retinal vasculature than those that only completed first round treatment. Repeated treatment with KRN633 decreased the anti-angiogenic effects but increased the immunoreactivity of α-smooth muscle actin in the pericytes on veins and capillaries. α-Smooth muscle actin expression was inversely correlated to anti-angiogenic effects. Overall, these results revealed that repeated interruption of VEGF receptor signaling pathway altered the phenotypes of endothelial cells and pericytes and induced anti-VEGF drug resistance. Therefore, careful follow-up is necessary when using anti-VEGF drugs to treat abnormal angiogenesis-associated ocular diseases.

Drug-Induced Mitochondrial Disruption in the Blood-Brain Barrier Cells: Overlooked Player in Drug Safety Evaluation.

The blood-brain barrier (BBB) is based on the unique pattern of the microvasculature of the central nervous system (CNS), which controls the transport of molecules between the CNS and the blood. The blood-brain barrier is mainly composed of endothelial cells, pericytes, and basement membrane, as well as the astrocytes and immune cells as perivascular macrophages and microglial cells. The dysfunction of this barrier can cause serious neuronal disorders due to the transport of hazardous molecules and immune cells to the CNS. Mitochondria plays a major role in cellular homeostasis in terms of health and disease. This review evaluated the published data about the effect of the drugs on the cells of BBB. Only seven articles were found that considered the effect of drugs on the barrier endothelial cells and mitochondria via different assays. Further studies are recommended to evaluate the impact of used medications on BBB cell bioenergetics. Also, the effect of the newly studied pharmaceutical agents on the BBB bioenergetics should be included within their safety profile studies.

Extragonadal function of follicle-stimulating hormone: Evidence for a role in endothelial physiology and dysfunction

AimsFollicle-stimulating hormone (FSH) plays a fundamental role in reproduction stimulating ovarian folliculogenesis, Sertoli cells function and spermatogenesis. However, the recent identification of FSH receptor (FSHR) also in extra-gonadal tissues has suggested that FSH activity may not be limited only to fertility regulation, with conflicting results on the possible role of FSH in endothelial cells. The aim of this study was to investigate FSH role on endothelial function in Human Umbilical Vein Endothelial Cells (HUVECs). ResultsEndothelial Nitric oxide synthase (eNOS) expression, eNOS phosphorylation and Nitric Oxide (NO) production resulted increased after the stimulation of HUVEC with recombinant human FSH (rhFSH) at 3.6x103 ng/ml, with increasing Calcium release from intracellular stores. Furthermore, IP3 production increased after rhFSH stimulation despite PTX treatment and NFAT1 was observed prevalently in nucleus.We observed a statistical difference between untreated cells and cells stimulated with 0.36x103 ng/ml and between cells stimulated with 0.36x103 ng/ml and cells stimulated with 1.8x103 ng/ml at 4 and 8 h by Wound healing assay, respectively. Furthermore, a higher cellular permeability was observed in stimulated cells, with atypical VE-cadherin distribution, as well as filamentous actin. ConclusionsOur findings suggest that FSH at high concentrations elicits a signalling that could compromise the endothelial membrane. Indeed, VE-cadherin anomalies may severely affect the endothelial barrier, resulting in an increased membrane permeability. Although NO is an important vasodilatation factor, probably an excessive production could impact on endothelial functionality, partially explaining the increased risk of cardiovascular diseases in menopausal women and men with hypogonadism.

An endothelium membrane mimetic antithrombotic coating enables safer and longer extracorporeal membrane oxygenation application

Thrombosis and plasma leakage are two of the most frequent dysfunctions of polypropylene (PP) hollow fiber membrane (PPM) used in extracorporeal membrane oxygenation (ECMO) therapy. In this study, a superhydrophilic endothelial membrane mimetic coating (SEMMC) was constructed on polydopamine-polyethyleneimine pre-coated surfaces of the PPM oxygenator and its ECMO circuit to explore safer and more sustainable ECMO strategy. The SEMMC is fabricated by multi-point anchoring of a phosphorylcholine and carboxyl side chained copolymer (PMPCC) and grafting of heparin (Hep) to form PMPCC-Hep interface, which endows the membrane superior hemocompatibility and anticoagulation performances. Furthermore, the modified PPM reduces protein adsorption amount to less than 30 ng/cm2. More significantly, the PMPCC-Hep coated ECMO system extends the anti-leakage and non-clotting oxygenation period to more than 15 h in anticoagulant-free animal extracorporeal circulation, much better than the bare and conventional Hep coated ECMO systems with severe clots and plasma leakage in 4 h and 8 h, respectively. This SEMMC strategy of grafting bioactive heparin onto bioinert zwitterionic copolymer interface has great potential in developing safer and longer anticoagulant-free ECMO systems. Statement of significanceA superhydrophilic endothelial membrane mimetic coating was constructed on surfaces of polypropylene hollow fiber membrane (PPM) oxygenator and its ECMO circuit by multi-point anchoring of a phosphorylcholine and carboxyl side chain copolymer (PMPCC) and grafting of heparin (Hep). The strong antifouling nature of the PMPCC-Hep coating resists the adsorption of plasma bio-molecules, resulting in enhanced hemocompatibility and anti-leakage ability. The grafted heparin on the zwitterionic PMPCC interface exhibits superior anticoagulation property. More significantly, the PMPCC-Hep coating achieves an extracorporeal circulation in a pig model for at least 15 h without any systemic anticoagulant. This endothelial membrane mimetic anticoagulation strategy shows great potential for the development of safer and longer anticoagulant-free ECMO systems.

Mitochondrial function in lungs of rats with different susceptibilities to hyperoxia-induced acute lung injury.

Adult rats exposed to hyperoxia (>95% O2) die from respiratory failure in 60-72 h. However, rats preconditioned with >95% O2 for 48 h followed by 24 h in room air acquire tolerance of hyperoxia (H-T), whereas rats preconditioned with 60% O2 for 7 days become more susceptible (H-S). Our objective was to evaluate lung tissue mitochondrial bioenergetics in H-T and H-S rats. Bioenergetics was assessed in mitochondria isolated from lung tissue of H-T, H-S, and control rats. Expressions of complexes involved in oxidative phosphorylation (OxPhos) were measured in lung tissue homogenate. Pulmonary endothelial filtration coefficient (Kf) and tissue mitochondrial membrane potential (Δψm) were evaluated in isolated perfused lungs (IPLs). Results show that ADP-induced state 3 OxPhos capacity (Vmax) decreased in H-S mitochondria but increased in H-T. Δψm repolarization time following ADP-stimulated depolarization increased in H-S mitochondria. Complex I expression decreased in H-T (38%) and H-S (43%) lung homogenate, whereas complex V expression increased (70%) in H-T lung homogenate. Δψm is unchanged in H-S and H-T lungs, but complex II has a larger contribution to Δψm in H-S than H-T lungs. Kf increased in H-S, but not in H-T lungs. For H-T, increased complex V expression and Vmax counter the effect of the decrease in complex I expression on Δψm. A larger complex II contribution to Δψm along with decreased Vmax and increased Kf could make H-S rats more hyperoxia susceptible. Results are clinically relevant since ventilation with ≥60% O2 is often required for extended periods in patients with acute respiratory distress syndrome (ARDS).NEW & NOTEWORTHY We assessed lung tissue mitochondrial bioenergetics in rats with tolerance (H-T) or susceptibility (H-S) to hyperoxia-induced ARDS. Results from studies in isolated mitochondria, tissue homogenate, and isolated perfused lungs show that mitochondrial bioenergetics are differentially altered in H-T and H-S lungs suggesting a potential role for mitochondrial bioenergetics in hyperoxia-induced ARDS. Results are clinically relevant since hyperoxia exposure is a primary therapy for patients with ARDS, and differential sensitivity to hyperoxia surely occurs in humans.

Ying and Yang of Ceramide in the Vascular Endothelium.

Ceramides, a group of biologically active sphingolipids, have been described as the new cholesterol given strong evidence linking high plasma ceramide with endothelial damage, risk for early adverse cardiovascular events, and development of cardiometabolic disease. This relationship has sparked great interest in investigating therapeutic targets with the goal of suppressing ceramide formation. However, the growing data challenge this paradigm of ceramide as solely eliciting detrimental effects to the cardiovascular system. Studies show that ceramides are necessary for maintaining proper endothelial redox states, mechanosensation, and membrane integrity. Recent work in preclinical models and isolated human microvessels highlights that the loss of ceramide formation can in fact propagate vascular endothelial dysfunction. Here, we delve into these conflicting findings to evaluate how ceramide may be capable of exerting both beneficial and damaging effects within the vascular endothelium. We propose a unifying theory that while basal levels of ceramide in response to physiological stimuli are required for the production of vasoprotective metabolites such as S1P (sphingosine-1-phosphate), the chronic accumulation of ceramide can promote activation of pro-oxidative stress pathways in endothelial cells. Clinically, the evidence discussed here highlights the potential challenges associated with therapeutic suppression of ceramide formation as a means of reducing cardiovascular disease risk.

Endothelial protein disulfide isomerase A1 enhances membrane stiffness and platelet-endothelium interaction in hyperglycemia via SLC3A2 and LAMC1

BackgroundDiabetes carries an increased risk of cardiovascular disease and thromboembolic events. Upon endothelial dysfunction, platelets bind to endothelial cells to precipitate thrombus formation; however, it is unclear which surface proteins regulate platelet-endothelium interaction. We and others have shown that peri/epicellular protein disulfide isomerase A1 (pecPDI) influences the adhesion and migration of vascular cells. ObjectivesWe investigated whether pecPDI regulates adhesion-related molecules on the surface of endothelial cells and platelets that influence the binding of these cells in hyperglycemia. MethodsImmunofluorescence was used to assess platelet-endothelium interaction in vitro, cytoskeleton reorganization, and focal adhesions. Hydrogen peroxide production was assessed via Amplex Red assays (ThermoFisher Scientific). Cell biophysics was assessed using atomic force microscopy. Secreted proteins of interest were identified through proteomics (secretomics), and targets were knocked down using small interfering RNA. Protein disulfide isomerase A1 (PDI) contribution was assessed using whole-cell PDI or pecPDI inhibitors or small interfering RNA. ResultsPlatelets of healthy donors adhered more onto hyperglycemic human umbilical vein endothelial cells (HUVECs). Endothelial, but not platelet, pecPDI regulated this effect. Hyperglycemic HUVECs showed marked cytoskeleton reorganization, increased H2O2 production, and elongated focal adhesions. Indeed, hyperglycemic HUVECs were stiffer compared with normoglycemic cells. PDI and pecPDI inhibition reversed the abovementioned processes in hyperglycemic cells. A secretomics analysis revealed 8 proteins secreted in a PDI-dependent manner by hyperglycemic cells. Among these, we showed that genetic deletion of LAMC1 and SLC3A2 decreased platelet-endothelium interaction and did not potentiate the effects of PDI inhibitors. ConclusionEndothelial pecPDI regulates platelet-endothelium interaction in hyperglycemia through adhesion-related proteins and alterations in endothelial membrane biophysics.

Tropism-shifted AAV-PHP.eB-mediated bFGF gene therapy promotes varied neurorestoration after ischemic stroke in mice.

AAV-PHP.eB is an artificial adeno-associated virus (AAV) that crosses the blood-brain barrier and targets neurons more efficiently than other AAVs when administered systematically. While AAV-PHP.eB has been used in various disease models, its cellular tropism in cerebrovascular diseases remains unclear. In the present study, we aimed to elucidate the tropism of AAV-PHP.eB for different cell types in the brain in a mouse model of ischemic stroke and evaluate its effectiveness in mediating basic fibroblast growth factor (bFGF) gene therapy. Mice were injected intravenously with AAV-PHP.eB either 14 days prior to (pre-stroke) or 1 day following (post-stroke) transient middle cerebral artery occlusion. Notably, we observed a shift in tropism from neurons to endothelial cells with post-stroke administration of AAV-PHP.eB-mNeonGreen (mNG). This endothelial cell tropism correlated strongly with expression of the endothelial membrane receptor lymphocyte antigen 6 family member A (Ly6A). Furthermore, AAV-PHP.eB-mediated overexpression of bFGF markedly improved neurobehavioral outcomes and promoted long-term neurogenesis and angiogenesis post-ischemic stroke. Our findings underscore the significance of considering potential tropism shifts when utilizing AAV-PHP.eB-mediated gene therapy in neurological diseases and suggest a promising new strategy for bFGF gene therapy in stroke treatment.

Elevated cutaneous expression of stem cell factor in chronic spontaneous urticaria: a prospective cohort study.

Tissue expression of endothelial cell (EC) markers of microcirculatory changes in CSU is poorly investigated. to explore the expression of specific EC markers (stem cell factor (SCF), vascular endothelial growth factor (VEGF) and membrane attack complex (MAC)) in CSU-L and CSU-NL skin through immunohistochemistry (IHC) and in serum. Lesional (L) and non-lesional (NL) skin biopsies from CSU patients and HCs were studied for the IHC expression of SCF, VEGF and MAC in CSU patients (n = 23) and healthy controls (HCs, n = 9). In this population, we also investigated blood levels of VEGF and SCF. Patients were also assessed for clinical characteristics, disease activity, and markers of autoimmune CSU (aiCSU). Epidermal SCF reactivity was significantly higher in CSU-L skin compared to HC skin (p=0.026). In the dermis, SCF immunoreactivity was seen particularly on endothelial, perivascular and epithelial cells. In CSU-L skin, mean perivascular SCF stainings were significantly more intense compared to HCs (p<0.001). Furthermore, CSU-NL skin also showed significantly higher SCF stainings on dermal perivascular cells compared to HCs (p<0.001). CSU patients had the highest SCF immunoreactivity scores in the epidermis and/or on dermal ECs. These patients did not have significantly higher SCF serum levels. This is the first study to show elevated cutaneous expression of SCF in chronic spontaneous urticaria. These findings underline the potential therapeutic possibilities of anti-KIT antibodies in CSU treatment.

Formulation, Optimization and In-Vivo Characterization of Thermosensitive In-Situ Nasal Gel Loaded with Bacoside a for Treatment of Epilepsy.

The intranasal route has demonstrated superior systemic bioavailability due to its extensive surface area, the porous nature of the endothelial membrane, substantial blood flow, and circumvention of first-pass metabolism. In traditional medicinal practices, Bacopa monnieri, also known as Brahmi, is known for its benefits in enhancing cognitive functions and potential effects in epilepsy. This study aimed to develop and optimize a thermosensitive in-situ nasal gel for delivering Bacoside A, the principal active compound extracted from Bacopa monnieri. The formulation incorporated Poloxamer 407 as a thermogelling agent and HPMC K4M as the Mucoadhesive polymer. A 32-factorial design approach was employed for Optimization. Among the formulations. F7 exhibited the most efficient Ex-vivo permeation through the nasal mucosa, achieving 94.69 ± 2.54% permeation, and underwent a sol-gel transition at approximately 30.48°C. The study's factorial design revealed that gelling temperature and mucoadhesive strength were critical factors influencing performance. The potential of in-situ nasal Gel (Optimized Batch-F7) for the treatment of epilepsy was demonstrated in an in-vivo investigation using a PTZ-induced convulsion model. This formulation decreased both the occurrence and intensity of seizures. The optimized formulation F7 showcases significant promise as an effective nasal delivery system for Bacoside A, offering enhanced bioavailability and potentially increased efficacy in epilepsy treatment.

Deletion of tissue factor pathway inhibitor isoform beta or gamma, but not alpha, improves clotting in hemophilic mice

BackgroundTissue factor pathway inhibitor (TFPI) regulates tissue factor–triggered coagulation. Humans and mice express transcripts encoding for multidistributed (endothelial, platelet, and plasma) 3-Kunitz domain TFPIα and endothelial membrane–anchored 2-Kunitz TFPIβ. Mice express a third transcript, γ, that encodes plasma lipoprotein–associated 2-Kunitz TFPI. In humans, proteolysis of α and/or β produces plasma lipoprotein–associated 2-Kunitz TFPI at lower levels. In clinical trials, monoclonal antibodies that target all TFPI isoforms extend coagulation and correct bleeding in hemophilic patients but with some thrombosis risks. ObjectivesTo determine the impact of TFPI isoform-specific deletions on promoting clotting in hemophilic mice. MethodsEngineered TFPI isoform–specific, hemophilic (factor VIII-null) mice were evaluated for clotting. ResultsMice expressing any single TFPI isoform were healthy. Thrombin generation assays identified TFPIγ as the dominant anticoagulation isoform in mouse plasma. Hemostasis was assessed by serial bleeding times from a tail vein laceration. Repeatedly, after a clot forms, it was manually disrupted; the number of clots/disruptions occurring over a 15-minute period were reported. C57BL/6 and hemophilic mice clot on average 25.6 vs 5.4 times, respectively. On a hemophilia background, TFPIβ or TFPIγ-specific deletion improved clotting to 14.6 and 15.2 times, respectively (P < .0001). TFPIα-specific deletion was without impact, clotting 5.1 times. Heterozygous deletion of TFPIβ was effective, clotting 11.8 times (P < .0001). Heterozygous deletion of TFPIα or TFPIγ alone was ineffective, clotting 3.0 and 6.1 times, respectively, but heterozygous TFPIαγ deletion improved clotting to 11.2 times (P < .001). ConclusionIn hemophilic mice, endothelial TFPIβ and plasma γ-derived 2-Kunitz TFPI individually contribute more to bleeding than total TFPIα.

Versatile Design of NO-Generating Proteolipid Nanovesicles for Alleviating Vascular Injury.

Vascular injury is central to the pathogenesis and progression of cardiovascular diseases, however, fostering alternative strategies to alleviate vascular injury remains a persisting challenge. Given the central role of cell-derived nitric oxide (NO) in modulating the endogenous repair of vascular injury, NO-generating proteolipid nanovesicles (PLV-NO) are designed that recapitulate the cell-mimicking functions for vascular repair and replacement. Specifically, the proteolipid nanovesicles (PLV) are versatilely fabricated using membrane proteins derived from different types of cells, followed by the incorporation of NO-generating nanozymes capable of catalyzing endogenous donors to produce NO. Taking two vascular injury models, two types of PLV-NO are tailored to meet the individual requirements of targeted diseases using platelet membrane proteins and endothelial membrane proteins, respectively. The platelet-based PLV-NO (pPLV-NO) demonstrates its efficacy in targeted repair of a vascular endothelium injury model through systemic delivery. On the other hand, the endothelial cell (EC)-based PLV-NO (ePLV-NO) exhibits suppression of thrombosis when modified onto a locally transplanted small-diameter vascular graft (SDVG). The versatile design of PLV-NO may enable a promising therapeutic option for various vascular injury-evoked cardiovascular diseases.

Endothelial Membrane Potential Changes are Required for Propagated Vasodilation

Introduction: The coordination of blood flow in response to localized increases in metabolic activity is vital for tissue function. This adaptive response typically involves the release of vasoactive factors leading to local vasodilation. This local vasodilation must propagate upstream to proximal feed arterioles and arteries to ensure a coordinated increase in blood flow and adequate tissue perfusion. But the mechanisms underlying such propagated vasodilation remain to be fully understood. This study aims to identify the conduction pathway(s) that facilitate this response. The hypothesis is that endothelial membrane potential changes are required for propagated vasodilation to occur. Methods: To dissect the pathways involved in propagated vasodilation, we employed micropuffng techniques to locally apply vasoactive agents to precontracted rat 2nd order mesenteric arteries. Using a 50um tip micropipette activators were applied against a flowing perfusate, to limit the action of the drug, and the precise site of activators determined by co-application of the fluorophore sulforhodamine. Vasodilator responses were recorded at the site of application and at intervals upstream from the application site. Results: Local application of the muscarinic receptor agonist, acetylcholine (ACh), evoked robust vasodilator responses that propagated upstream from the site of application. The propagation of vasodilation terminated at the site of removal of a 100uM wide strip of endothelial cells. A high-potassium solution, which induces depolarization and ‘clamps’ the membrane potential, also inhibited propagated vasodilation. In contrast, the nitric oxide donor, sodium nitroprusside did not initiate propagated vasodilation in arteries with an intact endothelium. Conclusion: Collectively, these results show that the endothelium serves as the primary signalling pathways for propagated vasodilation. Furthermore, our results highlight the necessity of endothelial cell membrane potential change in the initiation of this vasodilator signal. These insights reinforce the endothelium’s pivotal role in coordinating vascular function. British Heart Foundation. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Aquaporins enriched in endothelial vacuole membrane regulate the diameters of microvasculature in hyperglycaemia.

In patients with diabetic microvascular complications, decreased perfusion or vascular occlusion, caused by reduced vascular diameter, is a common characteristic that will lead to insufficient blood supply. Yet, the regulatory mechanism and effective treatment approach remain elusive. Our initial findings revealed a notable decrease in the expression of human AQP1 in both diabetic human retina samples (49 healthy vs. 54 diabetic samples) and high-glucose-treated human retinal microvascular endothelial cells. Subsequently, our investigations unveiled a reduction in vascular diameter and compromised perfusion within zebrafish embryos subjected to high glucose treatment. Further analysis indicated a significant down-regulation of two aquaporins, aqp1a.1 and aqp8a.1, which are highly enriched in ECs and are notably responsive to hyperglycaemic conditions. Intriguingly, the loss of function of aqp1a.1 and/or aqp8a.1 resulted in a reduction of intersegmental vessel diameters, effectively mirroring the phenotype observed in the hyperglycaemic zebrafish model. The overexpression of aqp1a.1/aqp8a.1 in zebrafish ECs led to notable enlargement of microvascular diameters. Moreover, the reduced vessel diameters resulting from high-glucose treatment were effectively rescued by the overexpression of these aquaporins. Additionally, both aqp1a.1 and apq8a.1 were localized in the intracellular vacuoles in cultured ECs as well as the ECs of sprouting ISVs, and the loss of Aqps caused the reduction of those vacuoles, which was required for lumenization. Notably, while the loss of AQP1 did not impact EC differentiation from human stem cells, it significantly inhibited vascular formation in differentiated ECs. EC-enriched aquaporins regulate the diameter of blood vessels through an intracellular vacuole-mediated process under hyperglycaemic conditions. These findings collectively suggest that aquaporins expressed in ECs hold significant promise as potential targets for gene therapy aimed at addressing vascular perfusion defects associated with diabetes.

DEVELOPMENT OF NASAL PHARMACEUTICAL PRODUCTS FOR SYSTEMIC EXPOSURE: USE OF PERMEATION ENHANCERS TO ACHIEVE TARGET PRODUCT PROFILE

In recent years, special attention has been paid to non-invasive parenteral routes of administration, which are an alternative to injectable drugs, as well as other dosage forms whose effectiveness and safety may be compromised by the administration route. At the same time, there is a paradigm shift in relation to nasal dosage forms: they are no longer considered as primarily drugs exhibiting local effects, and an increasing number of nasal medici-nal products with systemic action are appearing on the market. There is a growing interest to the nasal delivery due to the possibility of maximizing the therapeutic potential of active substances that have proven themselves in clinical practice, for example, by quick targeted action to the site of the pathological process, increased bioavailability of the drug and reduced possible side effects. The nasal cavity as a site for systemic drug absorption has anatomical and physiological features that include a relatively large surface area, a porous endothelial basement membrane, a highly vascularized epithelial layer, high total blood flow, lack of first-pass metabolism, and easy access. Therefore, the development of a convenient (nasal) dosage form of the drug with a rapid onset of action is a promising approach. When developing a new pharmaceutical product, the factors that determine bioavaila-bility should be taken into account and addressed by appropriate formulation and delivery techniques: the use of mucoadhesive components or sub-stances that modulate mucociliary clearance, variation of the viscosity or osmolarity, or selection of an appropriate dosing device. Improving intrana-sal absorption is a promising direction for obtaining drugs with improved consumer properties. In addition to the functional properties of the formula-tions, safety aspects must also be taken into account. The article provides an analysis of the limiting factors for nasal preparations, strategies for in-creasing bioavailability, permeation enhancement mechanisms, and provides examples of the application of permeation enhancers used in developed and commercially available drugs. This review gives an idea on possible to use in nasal formulations permeation enhancers which have different mode of actions.

Endothelial PTP1B Deletion Promotes VWF Exocytosis and Venous Thromboinflammation.

Endothelial activation promotes the release of procoagulant extracellular vesicles and inflammatory mediators from specialized storage granules. Endothelial membrane exocytosis is controlled by phosphorylation. We hypothesized that the absence of PTP1B (protein tyrosine phosphatase 1B) in endothelial cells promotes venous thromboinflammation by triggering endothelial membrane fusion and exocytosis. Mice with inducible endothelial deletion of PTP1B (End.PTP1B-KO) underwent inferior vena cava ligation to induce stenosis and venous thrombosis. Primary endothelial cells from transgenic mice and human umbilical vein endothelial cells were used for mechanistic studies. Vascular ultrasound and histology showed significantly larger venous thrombi containing higher numbers of Ly6G (lymphocyte antigen 6 family member G)-positive neutrophils in mice with endothelial PTP1B deletion, and intravital microscopy confirmed the more pronounced neutrophil recruitment following inferior vena cava ligation. RT2 PCR profiler array and immunocytochemistry analysis revealed increased endothelial activation and adhesion molecule expression in primary End.PTP1B-KO endothelial cells, including CD62P (P-selectin) and VWF (von Willebrand factor). Pretreatment with the NF-κB (nuclear factor kappa B) kinase inhibitor BAY11-7082, antibodies neutralizing CD162 (P-selectin glycoprotein ligand-1) or VWF, or arginylglycylaspartic acid integrin-blocking peptides abolished the neutrophil adhesion to End.PTP1B-KO endothelial cells in vitro. Circulating levels of annexin V+ procoagulant endothelial CD62E+ (E-selectin) and neutrophil (Ly6G+) extracellular vesicles were also elevated in End.PTP1B-KO mice after inferior vena cava ligation. Higher plasma MPO (myeloperoxidase) and Cit-H3 (citrullinated histone-3) levels and neutrophil elastase activity indicated neutrophil activation and extracellular trap formation. Infusion of End.PTP1B-KO extracellular vesicles into C57BL/6J wild-type mice most prominently enhanced the recruitment of endogenous neutrophils, and this response was blunted in VWF-deficient mice or by VWF-blocking antibodies. Reduced PTP1B binding and tyrosine dephosphorylation of SNAP23 (synaptosome-associated protein 23) resulting in increased VWF exocytosis and neutrophil adhesion were identified as mechanisms, all of which could be restored by NF-κB kinase inhibition using BAY11-7082. Our findings show that endothelial PTP1B deletion promotes venous thromboinflammation by enhancing SNAP23 phosphorylation, endothelial VWF exocytosis, and neutrophil recruitment.

Alterations of the blood-brain barrier during aging.

The blood-brain barrier (BBB) is a complex and dynamic interface that regulates the exchange of molecules and cells between the blood and the central nervous system. It undergoes structural and functional changes during aging, which may compromise its integrity and contribute to the pathogenesis of neurodegenerative diseases. In recent years, advances in microscopy and high-throughput bioinformatics have allowed a more in-depth investigation of the aging mechanisms of BBB. This review summarizes age-related alterations of the BBB structure and function from six perspectives: endothelial cells, astrocytes, pericytes, basement membrane, microglia and perivascular macrophages, and fibroblasts, ranging from the molecular level to the human multi-system level. These basic components are essential for the proper functioning of the BBB. Recent imaging methods of BBB were also reviewed. Elucidation of age-associated BBB changes may offer insights into BBB homeostasis and may provide effective therapeutic strategies to protect it during aging.

Rat Model of Type 2 Diabetes Mellitus Recapitulates Human Disease in the Anterior Segment of the Eye

Changes in the anterior segment of the eye due to type 2 diabetes mellitus (T2DM) are not well-characterized, in part due to the lack of a reliable animal model. This study evaluated changes in the anterior segment, including crystalline lens health, corneal endothelial cell density, aqueous humor metabolites, and ciliary body vasculature, in a rat model of T2DM compared with human eyes. Male Sprague-Dawley rats were fed a high-fat diet (45% fat) or normal diet, and rats fed the high-fat diet were injected with streptozotocin intraperitoneally to generate a model of T2DM. Cataract formation and corneal endothelial cell density were assessed using microscopic analysis. Diabetes-related rat aqueous humor alterations were assessed using metabolomics screening. Transmission electron microscopy was used to assess qualitative ultrastructural changes ciliary process microvessels at the site of aqueous formation in the eyes of diabetic rats and humans. Eyes from the diabetic rats demonstrated cataracts, lower corneal endothelial cell densities, altered aqueous metabolites, and ciliary body ultrastructural changes, including vascular endothelial cell activation, pericyte degeneration, perivascular edema, and basement membrane reduplication. These findings recapitulated diabetic changes in human eyes. These results support the use of this model for studying ocular manifestations of T2DM and support a hypothesis postulating blood-aqueous barrier breakdown and vascular leakage at the ciliary body as a mechanism for diabetic anterior segment pathology.

In the Rat Hippocampus, Pilocarpine-Induced Status Epilepticus Is Associated with Reactive Glia and Concomitant Increased Expression of CD31, PDGFRβ, and Collagen IV in Endothelial Cells and Pericytes of the Blood-Brain Barrier.

In humans and animal models, temporal lobe epilepsy (TLE) is associated with reorganization of hippocampal neuronal networks, gliosis, neuroinflammation, and loss of integrity of the blood-brain barrier (BBB). More than 30% of epilepsies remain intractable, and characterization of the molecular mechanisms involved in BBB dysfunction is essential to the identification of new therapeutic strategies. In this work, we induced status epilepticus in rats through injection of the proconvulsant drug pilocarpine, which leads to TLE. Using RT-qPCR, double immunohistochemistry, and confocal imaging, we studied the regulation of reactive glia and vascular markers at different time points of epileptogenesis (latent phase-3, 7, and 14 days; chronic phase-1 and 3 months). In the hippocampus, increased expression of mRNA encoding the glial proteins GFAP and Iba1 confirmed neuroinflammatory status. We report for the first time the concomitant induction of the specific proteins CD31, PDGFRβ, and ColIV-which peak at the same time points as inflammation-in the endothelial cells, pericytes, and basement membrane of the BBB. The altered expression of these proteins occurs early in TLE, during the latent phase, suggesting that they could be associated with the early rupture and pathogenicity of the BBB that will contribute to the chronic phase of epilepsy.