Human Dermal Microvascular Endothelial Cells Research Articles

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

Ethnopharmacological relevanceSeveral 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 methodsThe 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. ResultsOpuntia 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. ConclusionsThe 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.

AMPK Protects against lps-induced endothelial hyperpermeability via activation of p38 mapk/hsp27 pathway and control of junctional proteins expression

Disruption of endothelial barrier integrity is a critical factor in the pathogenesis of sepsis. We have earlier reported that AICAR protects against sepsis-induced hyperpermeability through AMPK activation, which preserves cell-cell junctions. We aimed to investigate the molecular mechanisms underlying the beneficial action of AMPK activation on cell junctions by using a recently-described more potent AMPK-activator called 991. Human dermal microvascular endothelial cells (HDMECs) were incubated with 991 (0.1 μM, 1 hour) before LPS treatment (50 μg/ml, 6 hours). Cell permeability was achieved by testing streptavidin-peroxydase endothelial flux in transwell assays. Cytoskeletal rearrangement was tested through actin immunostaining and cellular junctions expression level was assessed by western blot. The specificity of 991 action was validated by siRNA transfection to genetically knockdown AMPKalpha1. We showed that 991 triggered AMPK activation in HDMECs and led to a drastic decrease of the LPS-induced hyperpermeability. In addition, formation of acting stress fibers along the cell periphery was enhanced, reinforcing tethering forces between neighbour cells and decreasing LPS-induced intercellular gaps. This was accompanied with higher phosphorylation level of p38 MAPK and HSP27. Transfection with AMPKalpha1 siRNA or treatment with the SB203580 p38 MAPK inhibitor, abrogated the effect of 991 on p38/HSP27 pathway, stress fibers formation and cell permeability. Furthermore, AMPKalpha1 deletion was also associated with lower expression levels of Zonula Occludens-1, VeCadherin and Connexin 43, three key components of interendothelial junctions. We conclude that AMPK activation protects the endothelial barrier function via activation of P38 MAPK/HSP27 pathway, which mediates cytoskeletal rearrangement. Our results also highlight AMPKalpha1 as a critical modulator of junctional proteins expression and integrity.

MiR-370 inhibits the angiogenic activity of endothelial cells by targeting smoothened (SMO) and bone morphogenetic protein (BMP)-2.

MicroRNAs (miRNAs) crucially modulate fundamental biologic processes such as angiogenesis. In the present study, we focused on the molecular function of miRNA-370-3p (miR-370) in regulating the angiogenic activity of endothelial cells (ECs). Transfection with miR-370 mimic (miR-370m) significantly inhibited the sprouting of human dermal microvascular EC (HDMEC) and HUVEC spheroids and mouse aortic rings, whereas miR-370 inhibitor (miR-370i) promoted sprout formation. Additional in vitro assays demonstrated the pleiotropic inhibitory effects of miR-370m on HDMEC proliferation, migration, and tube formation. Moreover, Matrigel plugs containing miR-370m-transfected HDMECs exhibited a reduced microvessel density after implantation into CD1 nude mice when compared with controls. In contrast, miR-370i exerted proangiogenic effects. Mechanistic analyses revealed that miR-370 directly targets smoothened (SMO) and down-regulates bone morphogenetic protein (BMP)-2 expression in HDMECs. Accordingly, inhibition of SMO by cyclopamine reversed miR-370i-induced HDMEC proliferation and migration. In addition, BMP-2 treatment counteracted miR-370m-suppressed tube formation of HDMECs, whereas blockade of BMP-2 with neutralizing antibody significantly inhibited miR-370i-induced tube formation. Taken together, these novel findings indicate that miR-370 is a potent inhibitor of angiogenesis, which directly targets SMO and BMP-2.-Gu, Y., Becker, V., Zhao, Y., Menger, M. D., Laschke, M. W. miR-370 inhibits the angiogenic activity of endothelial cells by targeting smoothened (SMO) and bone morphogenetic protein (BMP)-2.

Intracellular availability of poorly soluble drugs from lipid nanocapsules

Lipid nanocapsules (LNCs) are extensively used as drug carrier systems, due to their small size distribution, biocompatibility and ease of preparation. They are especially useful for lipophilic drugs to overcome physicochemical constraints that limit their efficacy, such as low solubility in aqueous media. The aim of this work was to investigate the relationship between the intracellular availability of poorly soluble drugs delivered via LNCs and their biological efficacy in cells in vitro. Cyclosporin A (CsA) with a logPOct = 4.3 (Lucangioli et al., 2003) and Itraconazole (It) with a logPOct = 6.2 (Bhardwaj et al., 2013) served as model lipophilic compounds, as they are highly promising candidates for the treatment of neovascular ocular diseases. Due to their lipophilic properties and the resulting preference for the oily core of LNCs, high encapsulation efficiencies were achieved. Drug-loaded LNCs with particle sizes around 50 nm were grafted with an αvβ3 integrin ligand (RGD) to optimize cellular uptake by human dermal microvascular endothelial cells. Even though RGD-LNCs showed excellent internalization, they exhibited insufficient inhibitory effects in vitro regarding endothelial cell proliferation, vascular endothelial growth factor expression, and tube formation in contrast to free drugs. This loss of efficacy could be explained by negligible intracellular availability of the poorly soluble drugs from LNCs.

Proteomic Analysis of Endothelial Cells Exposed to Ultrasmall Nanoparticles Reveals Disruption in Paracellular and Transcellular Transport.

The large interactive surfaces of nanoparticles (NPs) increase the opportunities to develop NPs for vascular targeting. Proteomic analysis of endothelial cells exposed to NPs reveals the cellular response and turns the focus into the impairment of the endothelial permeability. Here, quantitative proteomics and transcriptome sequencing are combined to evaluate the effects of exposure to sub-lethal concentrations of TiO2 -USNPs and TiO2 -NPs on human dermal microvascular endothelial cells. Endothelial cells react to preserve the semi-permeable properties that are essential for vascular tissue fluid homeostasis, vascular development, and angiogenesis. The main impact of the exposure was alteration of functional complexes involved in cell adhesion, vesicular transport, and cytoskeletal structure. Those are the core cellular structures that are linked to the permeability and the integrity of the endothelial tissue. Moreover, the extracellular proteins uptake along wih the NPs into the endothelial cells escape the lysosomal degradation pathway. These findings improve the understanding of the interaction of NPs with endothelial cell. The effects of the studied NPs modulating cell-cell adhesion and vesicular transport can help to evaluate the distribution of NPs via intravenous administration.

IPSC-Derived Brain Endothelium Exhibits Stable, Long-Term Barrier Function in Perfused Hydrogel Scaffolds.

SummaryThere is a profound need for functional, biomimetic in vitro tissue constructs of the human blood-brain barrier and neurovascular unit (NVU) to model diseases and identify therapeutic interventions. Here, we show that induced pluripotent stem cell (iPSC)-derived human brain microvascular endothelial cells (BMECs) exhibit robust barrier functionality when cultured in 3D channels within gelatin hydrogels. We determined that BMECs cultured in 3D under perfusion conditions were 10–100 times less permeable to sodium fluorescein, 3 kDa dextran, and albumin relative to human umbilical vein endothelial cell and human dermal microvascular endothelial cell controls, and the BMECs maintained barrier function for up to 21 days. Analysis of cell-cell junctions revealed expression patterns supporting barrier formation. Finally, efflux transporter activity was maintained over 3 weeks of perfused culture. Taken together, this work lays the foundation for development of a representative 3D in vitro model of the human NVU constructed from iPSCs.

The antitumor efficacy of monomeric disintegrin obtustatin in S-180 sarcoma mouse model.

Obtustatin, isolated from the Levantine Viper snake venom (Macrovipera lebetina obtusa -MLO), is the shortest known monomeric disintegrin shown to specifically inhibit the binding of the α1β1 integrin to collagen IV. Its oncostatic effect is due to the inhibition of angiogenesis, likely through α1β1 integrin inhibition in endothelial cells. To explore the therapeutic potential of obtustatin, we studied its effect in S-180 sarcoma-bearing mice model in vivo as well as in human dermal microvascular endothelial cells (HMVEC-D) in vitro, and tested anti-angiogenic activity in vivo using the chick embryo chorioallantoic membrane assay (CAM assay). Our in vivo results show that obtustatin inhibits tumour growth by 33%. The expression of vascular endothelial growth factor (VEGF) increased after treatment with obtustatin, but the level of expression of caspase 8 did not change. In addition, our results demonstrate that obtustatin inhibits FGF2-induced angiogenesis in the CAM assay. Our in vitro results show that obtustatin does not exhibit cytotoxic activity in HMVEC-D cells in comparison to in vivo results. Thus, our findings disclose that obtustatin might be a potential candidate for the treatment of sarcoma in vivo with low toxicity.

Transcriptional profiles of cytokines and chemokines reveal important pro-inflammatory response from endothelial cells during Orientia tsutsugamushi infection

Endothelial cells (EC) are key targets during Orientia tsutsugamushi infection. Knowledge of the pro-inflammatory response against O. tsutsugamushi by ECs is limited. The aim of the present study was to characterize the pro-inflammatory transcriptional response during the first 24 h of infection of the human dermal microvascular endothelial cell line with O. tsutsugamushi Karp by examining five-time points. The transcriptional profiles of 84 genes including cytokines, chemokines, growth factors, and TNF receptor superfamily genes were studied using a RT-PCR array. We identified 40 of the 84 genes that were up or down modulated during the early O. tsutsugamushi infection that differed remarkably from genes of non-infected cells. The modulated genes included: the interleukins (IL-1α/β, IL-4, IL-6, IL-7, IL-10, IL-11, IL-18, and IL-24), chemokines (CXCL8, CCL2/MCP1, CCL5/RANTES, and CCL17), growth factors (NODAL, CNTF, and CSF2/GM-CSF), and TNFSF13B. IL-1β, IL-4, and IL-11 were highly induced at one hour post infection, whereas, CCL17 was profoundly up-regulated and IFNα2 was greatly down-regulated during the entire 24-hour time course. These results provide insight into the early pro-inflammatory response of endothelial cells to O. tsutsugamushi infection and indicate their potential role in the pathophysiology of the host's initial response to O. tsutsugamushi infection.

Engineering vasculature: Architectural effects on microcapillary-like structure self-assembly.

One of the greatest obstacles to clinical translation of bone tissue engineering is the inability to effectively and efficiently vascularize scaffolds. The goal of this work was to explore systematically whether architecture, at a scale of hundreds of microns, can be used to direct the growth of microcapillary-like structures into the core of scaffolds. Biphasic bioceramic patterned architectures were produced using silicone molds of 3D printed parts. Grooves and ridges were designed to have widths of 330 μm and 660 μm, with periodicities respectively of 1240 μm and 630 μm. Groove depth was varied between 150 μm and 585 μm. Co-cultures of human dermal microvascular endothelial cells (HDMECs) and human osteoblasts (hOBs) were used to grow microcapillary-like structures on substrates. Bioceramic architecture was found to significantly affect microcapillary-like structure location and orientation. Microcapillary-like structures were found to form predominantly in grooves or between convexities. For all patterned samples, the CD31 (endothelial cell marker) signal was at least 2.5 times higher along grooves versus perpendicular to grooves. In addition, the average signal was at least two times higher within grooves than outside grooves for all samples. Grooves with a width of 330 μm and a depth of 300 μm resulted in the formation of individual, highly aligned microcapillary-like structures with lengths around 5 mm. Extensive literature has focused on the role of nano- and micro-topography (on the scale below tens of microns) on cellular response. However, the idea that architecture at a scale much larger than a cell could be used to modulate angiogenesis has not been systematically investigated. This work shows the crucial influence of architecture on microcapillary-like structure self-assembly at the scale of hundreds of microns. Elucidating the precise correspondence between architecture and microcapillary-like structure organization will ultimately allow the engineering of microvasculature by tuning local scaffold design to achieve desirable microvessel properties.

Botulinum Toxin Type A Attenuates Apoptosis in Human Dermal Microvascular Endothelial Cells Exposed to an In Vitro Model of Ischemia/Reperfusion Injury

BackgroundBotulinum toxin type A (BTXA) has been reported to increase survival of critically ischemic skin flaps; however, the effect of BTXA in human dermal microvascular endothelial cells (HDMECs) remains to be investigated. This study aimed to investigate the protective effect of BTXA in HDMECs exposed to an in vitro model of ischemia/reperfusion injury. MethodsHDMECs were isolated from human upper eyelid tissue and were randomly divided into 3 groups: 1. control group: culture under normoxic conditions (95% air and 5% CO2); 2. hypoxia/reoxygenation (H/R) group: culture in a hypoxic incubator (94% N2 + 5% CO2 + 5% O2) for 8 hours, followed by culture in saturated aerobic culture medium for 24 hours; and 3. BTXA group: treatment with BTXA for 12 hours before exposure to hypoxic conditions. Flow cytometry was used to analyze the apoptosis of HDMECs, and western blotting was used to detect the expression of apoptosis-related proteins. ResultsH/R leads to severe injury in HDMECs, as evidenced by an increase in the percentage of apoptosis and an increase in expression of apoptosis-related proteins (Bax, cleaved-caspase-3, and cytochrome C). Moreover, H/R results in a decrease in expression of anti-apoptotic protein (Bcl-2), which can be significantly attenuated with BTXA treatment. ConclusionBTXA protects against apoptosis in HDMECs exposed to an in vitro model of H/R-induced injury.

Magnesium lithospermate B protects the endothelium from inflammation-induced dysfunction through activation of Nrf2 pathway.

Magnesium lithospermate B (MLB) is an active component of Salvia miltiorrhiza Radix, a traditional Chinese herb used in treating cardiovascular diseases. In this study, we investigated the protective effects of MLB against inflammation-induced endothelial dysfunction in vitro and in vivo, and the underlying mechanisms. Endothelial dysfunction was induced in human dermal microvascular endothelial cells (HMEC-1) in vitro by lipopolysaccharide (LPS, 1 μg/mL). We showed that pretreatment with MLB (10-100 μM) dose-dependently inhibited LPS-induced upregulation of inflammatory cytokines ICAM1, VCAM1, and TNFα, which contributed to reduced leukocytes adhesion and attenuation of endothelial hyperpermeability in HMEC-1 cells. SD rats were injected with LPS (10 mg/kg, ip) to induce endothelial dysfunction in vivo. We showed that pretreatment with MLB (25-100 mg/kg, ip) dose-dependently restored LPS-impaired endothelial-dependent vasodilation in superior mesenteric artery (SMA), attenuated leukocyte adhesion in mesenteric venules and decreased vascular leakage in the lungs. We further elucidated the mechanisms underlying the protective effects of MLB, and revealed that MLB pretreatment inhibited NF-κB activation through inhibition of IκBα degradation and subsequent phosphorylation of NF-κB p65 in vitro and in vivo. In HMEC-1 cells, MLB pretreatment activated the nuclear factor erythroid-2-related factor 2 (Nrf2) pathway. Knockdown of Nrf2 with siRNA abolished the inhibitory effects of MLB on IκBα degradation and ICAM1 up-regulation, which were mimicked by PKC inhibition (Gö6983) or PI3K/Akt inhibition (LY294002). In summary, our results demonstrate that MLB inhibits NF-κB activation through PKC- and PI3K/Akt-mediated Nrf2 activation in HMEC-1 cells and protects against LPS-induced endothelial dysfunction in murine model of acute inflammation.

Isolation and Culture of Human Dermal Microvascular Endothelial Cells.

Primary endothelial cells are needed for angiogenesis studies, and more particularly in the field of tissue engineering, to engineer pre-vascularized tissues. Investigations often use human umbilical vein endothelial cells due to their extensive characterization, but also because they are easy to obtain and isolate. An alternative is the use of human dermal microvascular endothelial cells, more representative of adult skin angiogenesis and vascularization processes. This chapter presents a detailed methodology to isolate and culture microvascular endothelial cells from skin biopsies based on enzymatic digestion and mechanical extraction.

Large 3D bioprinted tissue: Heterogeneous perfusion and vascularization

Large bioprinted tissues (dm3) are more and more accessible but their in vitro culture and maturation conditions stay an unchartered territory. In the present report, we aim to present a preliminary study of endothelialized large bioprinted tissues (fibroblast and human dermal microvascular endothelial cells) maturation using silicone 3D printed perfusion system (bioreactor). Computational Fluid Dynamics (CFD) simulation was used to relate the theoretical culture medium flow path within the large bioprinted tissue with the actual tissue morphology and composition, obtained through histological observations. The obtained results demonstrate the positive impact of using dynamic maturation conditions (300 mL/h culture medium flow rate) on the extracellular matrix production and the conservation of the large bioprinted tissue internal geometry. Clear differences between static and dynamic culture conditions were herein found. Finally, typical microvascular organization, composed of human dermal microvascular endothelial cells organized around an open lumen, were found within the large bioprinted tissue.

MicroRNA-Regulated Rickettsial Invasion into Host Endothelium via Fibroblast Growth Factor 2 and Its Receptor FGFR1.

Microvascular endothelial cells (ECs) represent the primary target cells during human rickettsioses and respond to infection via the activation of immediate–early signaling cascades and the resultant induction of gene expression. As small noncoding RNAs dispersed throughout the genome, microRNAs (miRNAs) regulate gene expression post-transcriptionally to govern a wide range of biological processes. Based on our recent findings demonstrating the involvement of fibroblast growth factor receptor 1 (FGFR1) in facilitating rickettsial invasion into host cells and published reports suggesting miR-424 and miR-503 as regulators of FGF2/FGFR1, we measured the expression of miR-424 and miR-503 during R. conorii infection of human dermal microvascular endothelial cells (HMECs). Our results revealed a significant decrease in miR-424 and miR-503 expression in apparent correlation with increased expression of FGF2 and FGFR1. Considering the established phenomenon of endothelial heterogeneity and pulmonary and cerebral edema as the prominent pathogenic features of rickettsial infections, and significant pathogen burden in the lungs and brain in established mouse models of disease, we next quantified miR-424 and miR-503 expression in pulmonary and cerebral microvascular ECs. Again, R. conorii infection dramatically downregulated both miRNAs in these tissue-specific ECs as early as 30 min post-infection in correlation with higher FGF2/FGFR1 expression. Changes in the expression of both miRNAs and FGF2/FGFR1 were next confirmed in a mouse model of R. conorii infection. Furthermore, miR-424 overexpression via transfection of a mimic into host ECs reduced the expression of FGF2/FGFR1 and gave a corresponding decrease in R. conorii invasion, while an inhibitor of miR-424 had the expected opposite effect. Together, these findings implicate the rickettsial manipulation of host gene expression via regulatory miRNAs to ensure efficient cellular entry as the critical requirement to establish intracellular infection.

Dengue virus or NS1 protein induces trans-endothelial cell permeability associated with VE-Cadherin and RhoA phosphorylation in HMEC-1 cells preventable by Angiopoietin-1.

A transient increase in trans-endothelial cell permeability in dengue patients leads to vascular leakage and shock syndrome. Here, we analysed the molecular mechanisms that cause permeability changes in human dermal microvascular endothelial cells (HMEC-1) using a direct dengue virus (DENV) infection model or treatment with NS1, a secreted DENV non-structural protein. In HMEC-1 cells, both treatments increase permeability with a concordant increase in the secretion of angiopoietin-2 (Ang-2). There is phosphorylation and loss of the junction protein VE-Cadherin from the inter-endothelial cell junctions and phosphorylation of RhoA. Direct virus infection results in activation of Src by phosphorylation, whereas NS1 treatment alone does not lead to Src activation. Furthermore, treatment with recombinant Ang-1, a physiological antagonist of Ang-2, prevents Ang-2 release, VE-Cadherin phosphorylation and internalization, and phosphorylation of RhoA and Src, resulting in restoration of barrier function. The permeability increase could also be prevented by blocking the Ang1/2 signalling receptor, Tie-2, or using a Rho/ROCK-specific inhibitor. Dasatinib, a Src-family kinase (SFK) inhibitor that inhibits Src phosphorylation, prevents enhanced permeability induced by direct DENV infection whereas in NS1 protein-treated cells its effect is less significant. The results provide important insights on the mechanisms of increased trans-endothelial permeability in DENV infection, and suggest the therapeutic potential of using recombinant Ang-1 or targeting these key molecules to prevent vascular leakage in dengue.

GW29-e1089 Cadmium exposure-induced abnormal lipid metabolism in human dermal microvascular endothelial cells

GW29-e1089 Cadmium exposure-induced abnormal lipid metabolism in human dermal microvascular endothelial cells

Botulinum toxin type A induces protective autophagy in human dermal microvascular endothelial cells exposed to an in vitro model of ischemia/reperfusion injury.

Botulinum toxin type A (BTXA) has been reported to increase the survival of ischemic skin flaps; however, the exact mechanism underlying this effect remains unclear and needs to be further established. The present study aimed to elucidate whether autophagy caused by BTXA functions as a protection mechanism and to identify the mechanisms of its regulation by BTXA in human dermal microvascular endothelial cells (HDMECs) subjected to hypoxia/reoxygenation (H/R)-induced injury. HDMECs were harvested from the upper eyelid tissues of female blepharoplasty patients. HDMECs were exposed to BTXA treatment for 12 h and then subjected to hypoxia for 8 h, followed by reoxygenation for 24 h. Chloroquine diphosphate salt (CQ) was used as an autophagy inhibitor. H/R led to extreme injury to the HDMECs as indicated by the rise in the apoptosis rate, which was significantly attenuated by BTXA pretreatment. The outcomes demonstrated that H/R caused autophagy, as evidenced by a higher type II/type I ratio of light chain 3 (LC3), increased expression of Beclin-1 and increased autophagosome formation. BTXA enhanced autophagy and attenuated apoptosis in a dose-dependent manner, whereas CQ attenuated the BTXA antiapoptotic effects and inhibited the formation of autophagolysosomes, which caused clustering of the LC3-II in cells. In conclusion, autophagy promoted by BTXA serves as a potential protective effect on ischemia/reperfusion injury.

Improved vasculogenesis and bone matrix formation through coculture of endothelial cells and stem cells in tissue‐specific methacryloyl gelatin‐based hydrogels

The coculture of osteogenic and angiogenic cells and the resulting paracrine signaling via soluble factors are supposed to be crucial for successfully engineering vascularized bone tissue equivalents. In this study, a coculture system combining primary human adipose-derived stem cells (hASCs) and primary human dermal microvascular endothelial cells (HDMECs) within two types of hydrogels based on methacryloyl-modified gelatin (GM) as three-dimensional scaffolds was examined for its support of tissue specific cell functions. HDMECs, together with hASCs as supporting cells, were encapsulated in soft GM gels and were indirectly cocultured with hASCs encapsulated in stiffer GM hydrogels additionally containing methacrylate-modified hyaluronic acid and hydroxyapatite particles. After 14 days, the hASC in the stiffer gels (constituting the "bone gels") expressed matrix proteins like collagen type I and fibronectin, as well as bone-specific proteins osteopontin and alkaline phosphatase. After 14 days of coculture with HDMEC-laden hydrogels, the viscoelastic properties of the bone gels were significantly higher compared with the gels in monoculture. Within the soft vascularization gels, the formed capillary-like networks were significantly longer after 14 days of coculture than the structures in the control gels. In addition, the stability as well as the complexity of the vascular networks was significantly increased by coculture. We discussed and concluded that osteogenic and angiogenic signals from the culture media as well as from cocultured cell types, and tissue-specific hydrogel composition all contribute to stimulate the interplay between osteogenesis and angiogenesis in vitro and are a basis for engineering vascularized bone.

TWEAK/Fn14 Signals Mediate Burn Wound Repair

TWEAK acts by engaging with Fn14 to regulate inflammatory responses, fibrosis, and tissue remodeling, which are central in the repair processes of wounds. This study aims to explore the potential role of the TWEAK/Fn14 pathway in the healing of cutaneous burn wounds. Third-degree burns were introduced in wild-type and Fn14-deficient BALB/c mice, followed by evaluation of wound areas and histological changes. The downstream cytokines including growth factors were also examined in lesional skin. Moreover, human dermal microvascular endothelial cells and dermal fibroblasts were analyzed invitro upon TWEAK stimulation. The healing of burn wounds was delayed in Fn14-deficient mice and was accompanied by the suppression of inflammatory responses, growth factor production, and extracellular matrix synthesis. Moreover, TWEAK/Fn14 activation enhanced the migration and cytokine production of both dermal microvascular endothelial cells and dermal fibroblasts. TWEAK also facilitates the expression of α-SMA and palladin in dermal fibroblasts. Furthermore, transfection of Fn14 small interfering RNA abrogated such promotion effect of TWEAK on these cells. In conclusion, TWEAK/Fn14 signals mediate the healing of burn wounds, possibly involving TWEAK regulation of the function of resident cells.

CXCL13 produced by macrophages due to Fli1 deficiency may contribute to the development of tissue fibrosis, vasculopathy and immune activation in systemic sclerosis.

CXCL13, a chemokine for B cells, follicular T cells, T helper 17 cells, and regulatory T cells, is reported to contribute to the development of systemic sclerosis (SSc), reflecting aberrant activation of immune system. To better understand the role of CXCL13 in SSc, we investigated the influence of Fli1 deficiency, a potential predisposing factor of this disease, on CXCL13 expression and assessed the clinical correlation of serum CXCL13 levels by multivariate regression analysis. Haploinsufficient loss of Fli1 remarkably induced CXCL13 expression in murine peritoneal macrophages, while gene silencing of FLI1 did not affect the expression of CXCL13 in human dermal fibroblasts and human dermal microvascular endothelial cells. Serum CXCL13 levels were elevated in SSc patients compared with healthy controls and correlated positively with skin score and negatively with pulmonary function test results. SSc patients with elevated serum CXCL13 levels had longer disease duration, diffuse cutaneous involvement, interstitial lung disease (ILD), heart involvement, pulmonary arterial hypertension, Raynaud's phenomenon, pitting scars, digital ulcers, telangiectasia, and high serum IgG levels more frequently than the other patients. In particular, serum CXCL13 levels were associated with ILD and digital ulcers by multivariate regression analysis. Taken together, these results indicate that CXCL13 expression is upregulated by Fli1 deficiency in macrophages, potentially contributing to the development of tissue fibrosis, vasculopathy and immune activation in SSc, especially ILD and digital ulcers.