Cerebral uptake of microvesicles occurs in normocapnic but not hypocapnic passive hyperthermia in young healthy male adults.

Passive hyperthermia causes cerebral hypoperfusion primarily from heat-induced respiratory alkalosis. However, despite the cerebral hypoperfusion, it is possible that the mild alkalosis might help to attenuate cerebral inflammation. In this study, the cerebral exchange of extracellular vesicles (microvesicles), which are known to elicit pro-inflammatory responses when released in conditions of stress, were examined in hyperthermia with and without respiratory alkalosis. Ten healthy male adults were heated passively, using a warm water-perfused suit, up to core temperature + 2°C. Blood samples were taken from the radial artery and internal jugular bulb. Microvesicle concentrations were determined in platelet-poor plasma via cells expressing CD62E (activated endothelial cells), CD31+ /CD42b- (apoptotic endothelial cells), CD14 (monocytes) and CD45 (pan-leucocytes). Cerebral blood flow was measured via duplex ultrasound of the internal carotid and vertebral arteries to determine cerebral exchange kinetics. From baseline to poikilocapnic (alkalotic) hyperthermia, there was no change in microvesicle concentration from any cell origin measured (P-values all >0.05). However, when blood CO2 tension was normalized to baseline levels in hyperthermia, there was a marked increase in cerebral uptake of microvesicles expressing CD62E (P = 0.028), CD31+ /CD42b- (P = 0.003) and CD14 (P = 0.031) compared with baseline, corresponding to large increases in arterial but not jugular venous concentrations. In a subset of seven participants who underwent hypercapnia and hypocapnia in the absence of heating, there was no change in microvesicle concentrations or cerebral exchange, suggesting that hyperthermia potentiated the CO2 /pH-mediated cerebral uptake of microvesicles. These data provide insight into a potential beneficial role of respiratory alkalosis in heat stress. KEY POINTS: The hyperthermia-induced hyperventilatory response is observed in most humans, despite causing potentially harmful reductions in cerebral blood flow. We tested the hypothesis that the respiratory-induced alkalosis is associated with lower circulating microvesicle concentrations, specifically in the brain, despite the reductions in blood flow. At core temperature + 2°C with respiratory alkalosis, microvesicles derived from endothelial cells, monocytes and leucocytes were at concentrations similar to baseline in the arterial and cerebral venous circulation, with no changes in cross-brain microvesicle kinetics. However, when core temperature was increased by 2°C with CO2 /pH normalized to resting levels, there was a marked cerebral uptake of microvesicles derived from endothelial cells and monocytes. The CO2 /pH-mediated alteration in cerebral microvesicle uptake occurred only in hyperthermia. These new findings suggest that the heat-induced hyperventilatory response might serve a beneficial role by preventing potentially inflammatory microvesicle uptake in the brain.

Effects of sleep deprivation and 4-7-8 breathing control on heart rate variability, blood pressure, blood glucose, and endothelial function in healthy young adults.

This study investigated the effects of sleep deprivation on heart rate variability (HRV), blood pressure (BP), fasting blood glucose (FBG), and endothelial function as well as the immediate effects of 4-7-8 breathing control on HRV and BP. In total, 43 healthy participants aged 19-25 years were classified into two groups: Twenty two in the with sleep deprivation group and 21 in the without sleep deprivation (control) group. Resting heart rate (HR), BP, HRV, FBG, and endothelial function were examined. Subsequently, participants practiced 4-7-8 breathing control for six cycles/set for three sets interspersed between each set by 1-min normal breathing. Thereafter, the HR, BP, and HRV were immediately examined. The HRV, HR, and BP variables and FBG were not significantly different between the two groups. However, endothelial function was significantly lower in the sleep deprivation group than that in the control group (p < 0.05). In response to 4-7-8 breathing control, low- and very-low-frequency powers significantly decreased (p < 0.05), whereas high-frequency power significantly increased (p < 0.05) in the control group. Moreover, time domain, total power, and very-low-frequency power significantly decreased (p < 0.05) in the sleep deprivation group. Both groups had significantly decreased HR and systolic BP (p < 0.05). HRV, HR, and BP variables showed no significant differences between the groups. Healthy young adults with and without sleep deprivation may have similar HRV, BP, and FBG values. However, sleep deprivation may cause decreased endothelial function. Furthermore, 4-7-8 breathing control can help participants improve their HRV and BP, particularly in those without sleep deprivation.

GlomSpheres as a 3D co-culture spheroid model of the kidney glomerulus for rapid drug-screening.

The glomerulus is the filtration unit of the kidney. Injury to any component of this specialised structure leads to impaired filtration and eventually fibrosis and chronic kidney disease. Current two and three dimensional (2D and 3D) models that attempt to recreate structure and interplay between glomerular cells are imperfect. Most 2D models are simplistic and unrepresentative, and 3D organoid approaches are currently difficult to reproduce at scale and do not fit well with current industrial drug-screening approaches. Here we report a rapidly generated and highly reproducible 3D co-culture spheroid model (GlomSpheres), better demonstrating the specialised physical and molecular structure of a glomerulus. Co-cultured using a magnetic spheroid formation approach, conditionally immortalised (CI) human podocytes and glomerular endothelial cells (GEnCs) deposited mature, organized isoforms of collagen IV and Laminin. We demonstrate a dramatic upregulation of key podocyte (podocin, nephrin and podocalyxin) and GEnC (pecam-1) markers. Electron microscopy revealed podocyte foot process interdigitation and endothelial vessel formation. Incubation with pro-fibrotic agents (TGF-ß1, Adriamycin) induced extracellular matrix (ECM) dysregulation and podocyte loss, which were attenuated by the anti-fibrotic agent Nintedanib. Incubation with plasma from patients with kidney disease induced acute podocyte loss and ECM dysregulation relative to patient matched remission plasma, and Nintedanib reduced podocyte loss. Finally, we developed a rapid imaging approach to demonstrate the model's usefulness in higher throughput pharmaceutical screening. GlomSpheres therefore represent a robust, scalable, replacement for 2D in vitro glomerular disease models.

Inflammatory and Biomechanical Drivers of Endothelial-Interstitial Interactions in Calcific Aortic Valve Disease.

Calcific aortic valve disease is dramatically increasing in global burden, yet no therapy exists outside of prosthetic replacement. The increasing proportion of younger and more active patients mandates alternative therapies. Studies suggest a window of opportunity for biologically based diagnostics and therapeutics to alleviate or delay calcific aortic valve disease progression. Advancement, however, has been hampered by limited understanding of the complex mechanisms driving calcific aortic valve disease initiation and progression towards clinically relevant interventions.

Impairment of type H vessels by NOX2-mediated endothelial oxidative stress: critical mechanisms and therapeutic targets for bone fragility in streptozotocin-induced type 1 diabetic mice.

Rationale: Mechanisms underlying the compromised bone formation in type 1 diabetes mellitus (T1DM), which causes bone fragility and frequent fractures, remain poorly understood. Recent advances in organ-specific vascular endothelial cells (ECs) identify type H blood vessel injury in the bone, which actively direct osteogenesis, as a possible player. Methods: T1DM was induced in mice by streptozotocin (STZ) injection in two severity degrees. Bony endothelium, the coupling of angiogenesis and osteogenesis, and bone mass quality were evaluated. Insulin, antioxidants, and NADPH oxidase (NOX) inhibitors were administered to diabetic animals to investigate possible mechanisms and design therapeutic strategies. Results: T1DM in mice led to the holistic abnormality of the vascular system in the bone, especially type H vessels, resulting in the uncoupling of angiogenesis and osteogenesis and inhibition of bone formation. The severity of osteopathy was positively related to glycemic levels. These pathological changes were attenuated by early-started, but not late-started, insulin therapy. ECs in diabetic bones showed significantly higher levels of reactive oxygen species (ROS) and NOX 1 and 2. Impairments of bone vessels and bone mass were effectively ameliorated by treatment with anti-oxidants or NOX2 inhibitors, but not by a NOX1/4 inhibitor. GSK2795039 (GSK), a NOX2 inhibitor, significantly supplemented the insulin effect on the diabetic bone. Conclusions: Diabetic osteopathy could be a chronic microvascular complication of T1DM. The impairment of type H vessels by NOX2-mediated endothelial oxidative stress might be an important contributor that can serve as a therapeutic target for T1DM-induced osteopathy.

Salvianic Acid A Regulates High-Glucose-Treated Endothelial Progenitor Cell Dysfunction via the AKT/Endothelial Nitric Oxide Synthase (eNOS) Pathway.

BACKGROUND The primary cause of death in patients with diabetes mellitus (DM) is diabetic macroangiopathy, a complication that related to the function and number of endothelial progenitor cells (EPCs). Salvianic acid A (SAA) is a water-soluble active ingredient of Salvia miltiorrhiza, a traditional Chinese medicine used to treat cardiovascular diseases. The purpose of this study was to explore the effects of SAA on the function of rat EPCs cultured in vitro in a high-glucose environment. MATERIAL AND METHODS Bone marrow-derived EPCs from 40 Sprague-Dawley rats were identified by fluorescence staining. Cell viability, apoptosis, tube formation, lactated dehydrogenase (LDH) release, and nitric oxide (NO) production were detected by 3-[4,5-dimethylthylthiazol-2-yl]-2,5 diphenyltetrazolium bromide assay, flow cytometry, tube formation, LDH, and 3-amino,4-aminomethyl-2',7'-difluorescein, and diacetate assays, respectively. The expression levels of proteins were examined by western blotting. RESULTS Cultured EPCs showed a cobblestone morphology and positive expression of Dil-ac-LDL and FITC-UEA-1. High glucose impaired cell viability. Different concentrations of SAA had no significant effect on EPC viability. SAA reduced the apoptosis rate and LDH release, but promoted tube formation, viability, and NO production in high-glucose-treated EPCs. The ratios of p-AKT/AKT and p-eNOS/eNOS in high-glucose-treated EPCs were elevated by SAA. Phosphoinositide 3-kinase inhibitor LY294002 blocked the rescue effects of SAA on high-glucose-treated EPCs. CONCLUSIONS SAA protected EPCs against high-glucose-induced dysfunction via the AKT/eNOS pathway.

Effects of DA-9801 on the inflammation and apoptosis induced by angiotensin II in human dermal microvascular endothelial cells.

DA-9801, a plant-based drug used for the treatment of diabetic neuropathy, is known to improve angiotensin II (Ang II)-induced vascular endothelial cell dysfunction. However, the underlying mechanism is not fully understood. We aimed to determine whether the protective effect of DA-9801 against Ang II-induced endothelial cell dysfunction was mediated via inhibition of endothelial cell inflammation and apoptosis. Ang II-induced oxidative stress was attenuated by pretreatment of human dermal microvascular endothelial cells (HDMECs) with DA-9801. This prevented the Ang II-induced upregulation of NAD(P)H oxidase (the NOX4 and p22phox subunits) and reactive oxygen species. Further, pretreatment of HDMECs with DA-9801 ameliorated Ang II-mediated nuclear factor kappa B activity via prevention of the upregulation of extracellular signal-regulated kinase and p38 mitogen-activated protein kinase. It also decreased the Ang II-stimulated increase in inducible nitric oxide synthase (NOS) and decreased endothelial NOS protein expression. DA-9801 decreased Ang II-induced upregulation of intercellular adhesion molecule 1, vascular adhesion molecule, and E-selectin in HDMECs. Moreover, TUNEL and annexin V-FITC fluorescence staining for apoptosis and the activities of caspases 9, 7, and 3 decreased in HDMECs pretreated with DA-9801, indicating that the drug enhanced anti-apoptotic pathways. Thus, DA-9801 modulated Ang II-induced endothelial cell dysfunction via inflammatory and apoptotic pathways.

The impact of glucose exposure on bioenergetics and function in a cultured endothelial cell model and the implications for cardiovascular health in diabetes.

Cardiovascular disease is the primary driver of morbidity and mortality associated with diabetes. Hyperglycaemia is implicated in driving endothelial dysfunction that might underpin the link between diabetes and cardiovascular disease. This study was designed to determine the impact of chronic preconditioning of cells to hyperglycaemia and transient switching of cultured endothelial cells between hyper- and normo-glycaemic conditions on bioenergetic and functional parameters. Immortalised EA.hy926 endothelial cells were cultured through multiple passages under normoglycaemic (5.5 mM) or hyperglycaemic (25 mM) conditions. Cells were subsequently subjected (48 h) to continued normo- or hyperglycaemic exposure, or were switched to the alternative glycaemic condition, or to an intermediate glucose concentration (12.5 mM) and metabolic activity, together with key markers of function were measured. Cells habituated to hyperglycaemia were energetically quiescent. Functional activity, characterised by the measurement of nitric oxide, endothelin-1, tissue plasminogen activator and plasminogen activator inhibitor-1, was depressed by exposure to high glucose, with the reduction in nitric oxide production being the most notable. Function was more responsive to acute changes in extracellular glucose than were bioenergetic changes. We conclude that glucose is a key determinant of endothelial function. The study highlights the importance of chronic glucose exposure on cell phenotype and emphasises the need to pay close attention to glucose preconditioning in interpreting results under culture conditions.

miRNA/Target Gene Profile of Endothelial Cells Treated with Human Triglyceride-Rich Lipoproteins Obtained after a High-Fat Meal with Extra-Virgin Olive Oil or Sunflower Oil.

SCOPE: The effects of triglyceride-rich lipoproteins (TRLs) on the miRNA expression of endothelial cells, which are very involved in atherosclerosis, according to the type of diet are not known. METHODS AND RESULTS: The differences between the effects of TRLs isolated from blood of subjects after a high-fat meal with extra-virgin olive oil (EVOO) and sunflower oil (SO) on the microRNA-Seq profile related to atherosclerosis in human umbilical vein endothelial cells are analyzed. 28 upregulated microRNAs with EVOO-derived TRLs, which can regulate 22 genes related to atherosclerosis, are found. 21 upregulated microRNAs with SO-derived TRLs, which can regulate 20 genes related to atherosclerosis, are found. These microRNAs are mainly involved in angiogenesis, with a predominance of an anti-angiogenic effect with EVOO-derived TRLs. Other microRNAs upregulated with SO-derived TRLs are involved in cardiovascular diseases. Pathways for the target genes obtained from the upregulated microRNA with EVOO-derived TRLs are involved in lipid metabolism and inflammatory and defense response, while those with SO-derived TRLs are involved in lipid metabolic process. CONCLUSION: EVOO-derived TRLs seem to produce a more atheroprotective profile than SO-derived TRLs. This study provides alternative mechanisms on the protective role of EVOO against the atherogenic process through microRNA regulation in endothelial cells.

Increased monocyte-platelet aggregates and monocyte-endothelial adhesion in healthy individuals with vitamin D deficiency.

Vitamin D deficiency is a major public health problem worldwide, linked to several chronic diseases including cardiovascular diseases. While immunomodulatory effects of vitamin D on monocytes have been reported in cardiovascular and metabolic diseases, there is limited understanding on monocyte phenotype in healthy individuals with suboptimal vitamin D levels and without any clinical diseases. In this work, we performed label-free, microfluidic isolation of monocytes, and characterized their functional phenotype using flow cytometry and in vitro vascular models in healthy subjects with (n = 7) and without vitamin D deficiency (n = 16). Vitamin D deficient (VitD-Def) subjects (25(OH)D3 level < 26 ng/mL) expressed significant downregulation of vitamin D receptor (VDR) on monocytes as compared to controls (P < .0001), and VDR expression was well-associated with serum 25(OH)D3 levels. Increased monocyte-platelet aggregates (MPA), a marker for platelet activation, were also observed in VitD-Def subjects (P < .05) which suggests a pro-inflammatory monocyte phenotype. Monocyte adhesion to endothelial cells, an early-stage atherosclerosis event, was also higher in VitD-Def individuals, and inversely correlated to serum 25(OH)D3 level (P < .05). Taken together, these results indicate the pro-inflammatory state and atherogenic potential of monocytes in VitD-Def healthy subjects, and propound the use of vitamin D supplementation as a prospective immunomodulatory and anti-inflammatory therapy in atherosclerosis.

"War to the knife" against thromboinflammation to protect endothelial function of COVID-19 patients.

In this viewpoint, we summarize the relevance of thromboinflammation in COVID-19 and discuss potential mechanisms of endothelial injury as a key point for the development of lung and distant organ dysfunction, with a focus on direct viral infection and cytokine-mediated injury. Entanglement between inflammation and coagulation and resistance to heparin provide a rationale to consider other therapeutic approaches in order to preserve endothelial function and limit microthrombosis, especially in severe forms. These strategies include nebulized heparin, N-acetylcysteine, plasma exchange and/or fresh frozen plasma, plasma derivatives to increase the level of endogenous anticoagulants (tissue factor pathway inhibitor, activated protein C, thrombomodulin, antithrombin), dipyridamole, complement blockers, different types of stem cells, and extracellular vesicles. An integrated therapy including these drugs has the potential to improve outcomes in COVID-19.

Anti-angiogenic effect of EGHB010, a standardized herbal formula of Paeoniae radix and Glycyrrhizae radix.

EGHB010 is a standardized herbal formula of the rhizome mixture of Paeonia lactiflora Pallas and Glycyrrhiza uralensis Fisch. Neovascularization in the retina is a common pathophysiology of diabetic retinal microvasculopathy and exudative macular degeneration. In this study, we evaluated the inhibitory effects of EGHB010 on abnormal retinal angiogenesis in a hyperoxia-induced neovascular retinopathy model. Vascular endothelial growth factor (VEGF)-mediated vascular tube formation was assayed in human umbilical vascular endothelial cells (HUVECs). Experimental angiogenesis in the retinas was induced by exposing C57BL/6 pups to hyperoxic environment (75% oxygen) on postnatal day 7 (P7) and then returning them to normal oxygen pressure on P12. EGHB010 (50 and 100 mg/kg/day) was administered intraperitoneally for 5 days (P12 - P16). Retinal flat mounts were prepared to measure the extent of retinal neovascularization on P17. The incubation of HUVECs with EGHB010 (1-25 µg/mL) resulted in the inhibition of VEGF-mediated tube formation in a dose-dependent manner. EGHB010 at doses of 50 and 100 mg/kg/day inhibited the formation of retinal neovascular tufts by 31.15±2.28% and 59.83±2.92%, respectively. Together, our results indicate that EGHB010 is a potent anti-angiogenic agent and may have potential for the control of abnormal retinal vessel growth in patients with ischemic retinopathy.

Mechanism of angiogenesis promotion with Shexiang Baoxin Pills by regulating function and signaling pathway of endothelial cells through macrophages.

BACKGROUND AND AIMS: "Shexiang Baoxin Pill" (SBP), a commonly used traditional Chinese medicine, has been used to treat angina, myocardial infarction and coronary heart disease in China for thirty years. SBP has been proven to promote angiogenesis in a rat model of myocardial infarction (MI). The aim of the present study was to determine the pro-angiogenic effects and mechanism of SBP during inflammation or ischemic pathological conditions and elucidate its regulatory effects on endothelial cell function and signaling pathways mediated by macrophages. METHODS: We used a polyvinyl alcohol (PVA) sponge implantation mouse model as an inflammatory angiogenesis model and utilized a mouse femoral artery ligation model as a hind limb ischemia model. We also performed cell proliferation, cell migration and tubule formation in vitro experiments to assess the effects of SBP on endothelial cell function and signaling pathways by stimulating macrophage activity. RESULTS: The in vitro experiment results showed that SBP could significantly increase the expression of mRNAs and proteins associated with angiogenesis in endothelial cells by activating macrophages to release pro-angiogenic factors such as Vegf-a. Activation of macrophages by SBP eventually led to endothelial cell proliferation, migration and tubule formation and increased the expression of p-Akt and p-Erk1/2 proteins in the downstream PI3K/Akt and MAPK/Erk1/2 signaling pathways related to angiogenesis, respectively. The in vivo experiment results indicated that SBP had angiogenesis effects in both inflammatory and ischemic angiogenesis models with dose- and time-dependent effects. CONCLUSION: Shexiang Baoxin Pills can promote angiogenesis by activating macrophages to regulate endothelial cell function and signal transduction pathways.

Activation of heme oxygenase-1 by Ginkgo biloba extract differentially modulates endothelial and smooth muscle-like progenitor cells for vascular repair.

Vascular progenitors such as endothelial progenitor cells (EPCs) and smooth muscle-like progenitor cells (SMPCs) may play different roles in vascular repair. Ginkgo biloba extract (GBE) is an exogenous activator of heme oxygenase (HO)-1, which has been suggested to improve vascular repair; however, the detailed mechanisms have yet to be elucidated. This study aimed to investigate whether GBE can modulate different vascular progenitor cells by activating HO-1 for vascular repair. A bone marrow transplantation mouse model was used to evaluate the in vivo effects of GBE treatment on wire-injury induced neointimal hyperplasia, which is representative of impaired vascular repair. On day 14 of GBE treatment, the mice were subjected to wire injury of the femoral artery to identify vascular reendothelialization. Compared to the mice without treatment, neointimal hyperplasia was reduced in the mice that received GBE treatment for 28 days in a dose-dependent manner. Furthermore, GBE treatment increased bone marrow-derived EPCs, accelerated endothelial recovery, and reduced the number of SMPCs attached to vascular injury sites. The effects of GBE treatment on neointimal hyperplasia could be abolished by co-treatment with zinc protoporphyrin IX, an HO-1 inhibitor, suggesting the in vivo role of HO-1. In this in vitro study, treatment with GBE activated human early and late EPCs and suppressed SMPC migration. These effects were abolished by HO-1 siRNA and an HO-1 inhibitor. Furthermore, GBE induced the expression of HO-1 by activating PI3K/Akt/eNOS signaling in human late EPCs and via p38 pathways in SMPCs, suggesting that GBE can induce HO-1 in vitro through different molecular mechanisms in different vascular progenitor cells. Accordingly, GBE could activate early and late EPCs, suppress the migration of SMPCs, and improve in vivo vascular repair after mechanical injury by activating HO-1, suggesting the potential role of pharmacological HO-1 activators, such as GBE, for vascular protection in atherosclerotic diseases.

Selective Adhesion and Directional Migration of Endothelial Cells Guided by Cys-Ala-Gly Peptide Density Gradient on Antifouling Polymer Brushes.

Selective adhesion and directional migration of endothelial cells (ECs) on biomaterials is critical to realize the rapid endothelialization. In this study, a Cys-Ala-Gly (CAG) peptide density gradient is generated on homogeneous cell-resisting poly(2-hydroxyethyl methacrylate-co-glycidyl methacrylate) brushes by immersing the brushes in a complementary gradient solution of CAG and competitive mercapto-terminated methoxyl poly(ethylene glycol). The adhesion and spreading of smooth muscle cells (SMCs) is impaired effectively on the gradient surface. About six folds of adherent ECs over SMCs are achieved at the position (10 mm) of highest CAG density on the gradient surface in a co-culture condition. Due to the gradient cues, ECs migrate fastest with the best directionality of 86.7% at the middle of the gradient, leading to the maximum net displacement as well.

RESPONDING TO SENSORIMOTOR SUGGESTIONS: From Endothelial Nitric Oxide to the Functional Equivalence Between Imagery and Perception.

The reduced cerebellar gray matter (GM) volume observed in highly hypnotizable individuals (highs) is likely due to the excessive release of endothelial nitric oxide in the brain and could account for their behavioral (postural and visuomotor control) and physiological (paradoxical pain control after cerebellar anodal stimulation) characteristics. Reduced cerebellar GM can induce low inhibition of the cerebral cortex, thus stronger functional equivalence (FE) between imagery and perception and greater proneness to respond to sensorimotor suggestions. In fact, stronger FE suggested in highs by behavioral studies has been confirmed by topological data analysis of EEG signals recorded during sensorimotor and imagery tasks. The authors' hypothesis cannot be applied to obstructive suggestions likely sustained by mechanisms related to socio-cognitive factors, i.e., oxytocin availability.

Cilostazol ameliorates ischemia/reperfusion-induced tight junction disruption in brain endothelial cells by inhibiting endoplasmic reticulum stress.

Endoplasmic reticulum (ER) stress is essential for brain ischemia/reperfusion (I/R) injury. However, whether it contributes to I/R-induced blood-brain barrier (BBB) injury remains unclear. cilostazol exerts protective effects toward I/R-induced BBB injury, with unclear mechanisms. This study explored the potential role of ER stress in I/R-induced endothelial cell damage and determined whether the therapeutic potential of cilostazol, with respect to I/R-induced endothelial cell damage, is related to inhibition of ER stress. We found that exposing brain endothelial cells (bEnd.3) to oxygen-glucose deprivation/reoxygenation (OGD/R) significantly activated ER stress and diminished the barrier function of cell monolayers; treatment with the ER stress inhibitor 4-phenylbutyric acid (4-PBA) or cilostazol prevented OGD/R-induced ER stress and preserved barrier function. Furthermore, OGD/R induced the expression and secretion of matrix metalloproteinase-9 and nuclear translocation of phosphorylated NF-κB. These changes were partially reversed by 4-PBA or cilostazol treatment. In vivo, 4-PBA or cilostazol significantly attenuated I/R-induced ER stress and ameliorated Evans blue leakage and tight junction loss. These results demonstrate that I/R-induced ER stress participates in BBB disruption. Targeting ER stress could be a useful strategy to protect the BBB from ischemic stroke, and cilostazol is a promising therapeutic agent for this process.-Nan, D., Jin, H., Deng, J., Yu, W., Liu, R., Sun, W., Huang, Y. Cilostazol ameliorates ischemia/reperfusion-induced tight junction disruption in brain endothelial cells by inhibiting endoplasmic reticulum stress.

Calligonum comosum (Escanbil) extract exerts anti-angiogenic, anti-proliferative and anti-inflammatory effects on endometriotic lesions.

ETHNOPHARMACOLOGICAL RELEVANCE: Calligonum comosum is a desert plant that is applied in traditional folkloric medicine for the treatment of abnormally heavy or prolonged menstruation and menstrual cramps. Moreover, it has been suggested for the treatment of infertility-causing conditions. Its bioactive chemical constituents inhibit multiple processes, such as angiogenesis, inflammation and invasive tissue growth, which may be beneficial in the therapy of endometriosis. AIM OF THE STUDY: We investigated the effects of Calligonum comosum on the development of endometriotic lesions. MATERIALS AND METHODS: We evaluated the anti-angiogenic activity of Calligonum comosum ethyl acetate fraction (CCEAF) in different in vitro angiogenesis assays. Moreover, we surgically induced endometriotic lesions in BALB/c mice, which received 50 mg/kg Calligonum comosum total extract (CCTE) or vehicle (control) over 4 weeks. The growth, cyst formation, vascularization and immune cell infiltration of the lesions were assessed with high-resolution ultrasound imaging, caliper measurements, histology and immunohistochemistry. RESULTS: CCEAF doses of up to 10 µg/mL did not impair the viability of human dermal microvascular endothelial cells (HDMEC), but dose-dependently suppressed their migration, tube formation and sprouting, indicating a substantial anti-angiogenic effect of CCEAF. Furthermore, CCTE significantly inhibited the growth and cyst formation of developing murine endometriotic lesions when compared to vehicle-treated controls. This was associated with a reduced vascularization, cell proliferation and immune cell infiltration. CONCLUSIONS: Our findings show that Calligonum comosum targets multiple, fundamental processes in the pathogenesis of endometriosis, which may be beneficial for the treatment of this common gynecological disorder.

Natural dietary antioxidants containing flavonoids modulate keratinocytes physiology: In vitro tri-culture models.

ETHNOPHARMACOLOGICAL RELEVANCE: Several traditional medicinal herbs are widely used for dermatologic and cosmetic preparations. The beneficial skin repair activity is detected in various phases of wound-healing process, such as cell-cell, cell-matrix interactions or collagen synthesis. AIM OF THE STUDY: The study assessed the effects of Opuntia ficus-indica (L.) Mill. (Opuntia) and Milk Thistle (MT) (Silybum marianum (L.) Gaerth) on adult keratinocytes (HaCaT) functioning under basal condition or in the presence of mechanical damage (wounded cells). MATERIALS AND METHODS: The role of the natural compounds was tested on HaCaT grown in mono-culture and tri-culture configurations. In tri-cultures models, HaCaT were treated with the conditioned media (CM) obtained by Human Normal Dermal Fibroblast (NHDF) and Human Dermal Microvascular Endothelial cells (HMVEC) co-cultures. Specifically, were tested cell viability, oxidative stress mechanisms (cytokines release and lipid peroxidation) and cellular remodelling (growth factors release or metalloproteinase modulation). Moreover, the migratory potential of HaCaT was analysed by the use of wound healing in vitro assay. RESULTS: Opuntia and MT differently modified the metabolism (EGF, MMP-9), and the migratory properties of HaCaT both under physiological conditions or upon mechanical damage (wounded cells). Moreover, both compounds modulated HaCaT response to oxidative stress. The response to the natural compounds were modified, and in some cases potentiated, in tri-culture configuration systems. CONCLUSIONS: The data demonstrated that in vitro tri-culture approach is suitable to characterize the role of natural compounds on the complex communication between dermal-epidermal cellular components and microvascular endothelium. Specifically, Opuntia and MT are good alternatives to synthetic compounds in skin repair promotion.

Antisteatotic and antioxidant activities of Thymbra spicata L. extracts in hepatic and endothelial cells as in vitro models of non-alcoholic fatty liver disease.

ETHNOPHARMACOLOGICAL RELEVANCE: Thymbra spicata, a member of the Lamiaceae family, is native to eastern Mediterranean area. Leaves of this plant are rich in phenolic compounds and are a popular remedy of traditional medicine in Lebanon to prevent and/or counteract hyperlipidemia and hyperglycemia. AIM OF THE STUDY: To evaluate the antisteatotic and antioxidant activities of extracts from leaves of Thymbra spicata L. using in vitro models of non-alcoholic fatty liver disease (NAFLD), a leading cause of liver-related morbidity and mortality worldwide, for whom no effective treatments are still available. MATERIALS AND METHODS: Two different extracts from Thymbra spicata L. aerial parts were prepared using water (TW) or ethanol (TE) as solvent. Their chemical composition was characterized by gas and liquid chromatography coupled with mass spectrometry. Both extracts were tested on cultured hepatic and endothelial cells treated to mimic in vitro a multisistemic pathology such as NAFLD. We assayed the effects on lipid accumulation, free radical production, lipid peroxidation, cell migration. RESULTS: Both the total phenolic and the total flavonoid contents were higher in the ethanolic extract. Rosmarinic acid was the most abundant polyphenol in TW, while TE was richer in carvacrol. Our findings demonstrated that both extracts ameliorated lipid accumulation, oxidative stress and inflammation in the NAFLD cellular models. However, the aqueous extract was more effective to reduce hepatic steatosis, and the ethanolic extract had higheranti-oxidant potential and wound healing activity. CONCLUSIONS: T. spicata extracts could be promising bioactive products to develop natural therapeutic agents or dietary supplements to treat NAFLD and obesity-related metabolic disease. Our findings suggest that while the ethanolic extract might be used in preventing endothelium dysfunction, the aqueous extract would act better as lipid-lowering agent.