Dermal Endothelial Cells Research Articles

Studies on anti-inflammatory activity of herbal extract mixture using inflammation induced human keratinocytes (HaCaT) cells

Inflammation is a general name for reactions occurring in several types of tissue injuries, infections, or immunologic stimulation as a defense against foreign or altered Endogenous substances. This comprises of a series of changes of the terminal tissues, which tend to eliminate the injurious agents and to repair the damaged tissue. Chemokines are a family of small proteins secreted by various cell types that play a role in the immune cell infiltration into inflammatory or infectious sites [1, 2]. Skin inflammation is closely associated with the production of Th2 chemokines [3]. These chemokines released from keratinocytes bind to and attract CCR4-positive Th2 cells into inflammatory tissues. It was reported that GJBRH inhibited the production of inflammatory cytokines by dermal endothelial cells. This study aimed to investigate whether GJBRH has inhibitory effects on TNF-α and IFN-γ-induced Th2 chemokine production and to identify the underlying molecular mechanisms in HaCaT human keratinocyte cell line. The present study was planned to evaluate the Herbal Extract sample for anti inflammatory activity using human skin keratinocytes

Head and neck dermatitis is exacerbated by Malassezia furfur colonization, skin barrier disruption, and immune dysregulation.

Head and neck dermatitis (HND) is a refractory phenotype of atopic dermatitis (AD) and can be a therapeutic challenge due to lack of responsiveness to conventional treatments. Previous studies have suggested that the microbiome and fungiome may play a role in inducing HND, but the underlying pathogenic mechanisms remain unknown. This study aimed to determine the link between HND and fungiome and to examine the contribution of Malassezia furfur. To identify the effect of the sensitization status of M. furfur on HND, 312 patients diagnosed with AD were enrolled. To elucidate the mechanism underlying the effects of M. furfur, human keratinocytes and dermal endothelial cells were cultured with M. furfur and treated with Th2 cytokines. The downstream effects of various cytokines, including inflammation and angiogenesis, were investigated by real-time quantitative PCR. To identify the association between changes in lipid composition and M. furfur sensitization status, D-squame tape stripping was performed. Lipid composition was evaluated by focusing on ceramide species using liquid chromatography coupled with tandem mass spectrometry. Increased sensitization to M. furfur was observed in patients with HND. Additionally, sensitization to M. furfur was associated with increased disease severity in these patients. IL-4 treated human keratinocytes cultured with M. furfur produced significantly more VEGF, VEGFR, IL-31, and IL-33. IL-4/M. furfur co-cultured dermal endothelial cells exhibited significantly elevated VEGFR, TGF-β, TNF-α, and IL-1β levels. Stratum corneum lipid analysis revealed decreased levels of esterified omega-hydroxyacyl-sphingosine, indicating skin barrier dysfunction in HND. Finally, M. furfur growth was inhibited by the addition of these ceramides to culture media, while the growth of other microbiota, including Cutibacterium acnes, were not inhibited. Under decreased levels of ceramide in AD patients with HND, M. furfur would proliferate, which may enhance pro-inflammatory cytokine levels, angiogenesis, and tissue remodeling. Thus, it plays a central role in the pathogenesis of HND in AD.

Coacervate-Filled Lipid Vesicles for Protein Delivery.

Macromolecularly crowded coacervate is useful in protein delivery for tissue engineering and regenerative medicine. However, coacervate tends to aggregate easily, which impedes their application. Here, this work presents a method to prepare coacervate with enhanced stability. This work assembles phospholipids on the surface of a coacervate to form lipocoacervate (LipCo). The resultant LipCo possesses a discrete spherical structure with a coacervate interior and phospholipid outer shell. The size of LipCo does not change over the four-week observation window, whereas coacervate coalesced into one bulk phase within 30 min. This work uses vascular endothelial growth factor-C (VEGF-C) and fibroblast growth factor-2 (FGF-2) as examples to test LipCo's ability to maintain protein bioactivity. The in vitro lymphangiogenesis assay demonstrates that human dermal lymphatic endothelial cells (LECs) formed increased network of cord in VEGF-C and FGF-2 loaded LipCo group compared to free proteins and proteins loaded in coacervate. Overall, LipCo could serve as a protein delivery vehicle with improved colloidal stability.

Immune-interacting lymphatic endothelial subtype at capillary terminals drives lymphatic malformation.

Oncogenic mutations in PIK3CA, encoding p110α-PI3K, are a common cause of venous and lymphatic malformations. Vessel type-specific disease pathogenesis is poorly understood, hampering development of efficient therapies. Here, we reveal a new immune-interacting subtype of Ptx3-positive dermal lymphatic capillary endothelial cells (iLECs) that recruit pro-lymphangiogenic macrophages to promote progressive lymphatic overgrowth. Mouse model of Pik3caH1047R-driven vascular malformations showed that proliferation was induced in both venous and lymphatic ECs but sustained selectively in LECs of advanced lesions. Single-cell transcriptomics identified the iLEC population, residing at lymphatic capillary terminals of normal vasculature, that was expanded in Pik3caH1047R mice. Expression of pro-inflammatory genes, including monocyte/macrophage chemokine Ccl2, in Pik3caH1047R-iLECs was associated with recruitment of VEGF-C-producing macrophages. Macrophage depletion, CCL2 blockade, or anti-inflammatory COX-2 inhibition limited Pik3caH1047R-driven lymphangiogenesis. Thus, targeting the paracrine crosstalk involving iLECs and macrophages provides a new therapeutic opportunity for lymphatic malformations. Identification of iLECs further indicates that peripheral lymphatic vessels not only respond to but also actively orchestrate inflammatory processes.

Single-cell profiling reveals a potent role of quercetin in promoting hair regeneration.

Hair loss affects millions of people at some time in their life, and safe and efficient treatments for hair loss are a significant unmet medical need. We report that topical delivery of quercetin (Que) stimulates resting hair follicles to grow with rapid follicular keratinocyte proliferation and replenishes perifollicular microvasculature in mice. We construct dynamic single-cell transcriptome landscape over the course of hair regrowth and find that Que treatment stimulates the differentiation trajectory in the hair follicles and induces an angiogenic signature in dermal endothelial cells by activating HIF-1α in endothelial cells. Skin administration of a HIF-1α agonist partially recapitulates the pro-angiogenesis and hair-growing effects of Que. Together, these findings provide a molecular understanding for the efficacy of Que in hair regrowth, which underscores the translational potential of targeting the hair follicle niche as a strategy for regenerative medicine, and suggest a route of pharmacological intervention that may promote hair regrowth.

A High-Fat Diet in the Absence of Obesity Increases Lymphangiogenesis by Inducing VEGF-C in a Murine Lymphedema Model.

Many researchers have attempted to induce lymphangiogenesis for the treatment of lymphedema. However, most previous studies had limited clinical usefulness. A high-fat diet (HFD) increases serum β-hydroxybutyrate (β-OHB) levels, which can stimulate lymphangiogenesis. The authors hypothesized that an HFD will ameliorate lymphedema through enhanced lymphangiogenesis. The effects of β-OHB on the lymphangiogenic process in human dermal lymphatic endothelial cells were analyzed. A mouse tail lymphedema model was used to evaluate the effects of an HFD on lymphedema. Experimental mice were fed an HFD (45% kcal as fat, 20% as protein, and 35% as carbohydrates) for 4 weeks. Tail volume was measured using the truncated cone formula. Biopsy specimens were taken 6 weeks after surgical induction of lymphedema. In human dermal lymphatic endothelial cells, treatment with 20 mM of β-OHB increased cell viability ( P = 0.008), cell migration ( P = 0.011), tube formation ( P = 0.005), and VEGF-C mRNA and protein expression ( P < 0.001) compared with controls. HFD feeding decreased tail volume by 14.3% and fibrosis by 15.8% ( P = 0.027), and increased the lymphatic vessel density ( P = 0.022) and VEGF-C protein expression ( P = 0.005) compared with those of operated, standard chow diet-fed mice. The authors' findings demonstrated that β-OHB promoted lymphatic endothelial cell function and increased VEGF-C mRNA and protein expression. When mice with tail lymphedema were fed an HFD, volume and fibrosis of the tail decreased. Therefore, the authors' findings suggest that an HFD can be a successful novel dietary approach to treating lymphedema. Lymphatic regeneration after vascularized lymph node transfer can be augmented when a high-fat diet is used in conjunction with vascularized lymph node transfer.

MiR-31-5p-Modified RAW 264.7 Macrophages Affect Profibrotic Phenotype of Lymphatic Endothelial Cells In Vitro.

Cardiac lymphatic vessel (LyV) remodeling as a contributor to heart failure has not been extensively evaluated in metabolic syndrome (MetS). Our studies have shown structural changes in cardiac LyV in MetS that contribute to the development of edema and lead to myocardial fibrosis. Tissue macrophages may affect LyV via secretion of various substances, including noncoding RNAs. The aim of the study was to evaluate the influence of macrophages modified by miR-31-5p, a molecule that regulates fibrosis and lymphangiogenesis, on lymphatic endothelial cells (LECs) in vitro. The experiments were carried out on the RAW 264.7 macrophage cell line and primary dermal lymphatic endothelial cells. RAW 264.7 macrophages were transfected with miR-31-5p and supernatant from this culture was used for LEC stimulation. mRNA expression levels for genes associated with lymphangiogenesis and fibrosis were measured with qRT-PCR. Selected results were confirmed with ELISA or Western blotting. miR-31-5p-modified RAW 264.7 macrophages secreted increased amounts of VEGF-C and TGF-β and a decreased amount of IGF-1. The supernatant from miR-31-5p-modified RAW 264.7 downregulated the mRNA expression for genes regulating endothelial-to-mesenchymal transition (EndoMT) and fibrosis in LECs. Our results suggest that macrophages under the influence of miR-31-5p show the potential to inhibit LEC-dependent fibrosis. However, more studies are needed to confirm this effect in vivo.

Bone Marrow Endothelial Cells Increase Prostate Cancer Cell Apoptosis in 3D Triculture Model of Reactive Stroma.

Simple SummaryProstate cancer (PCa) metastasizes preferentially to the bone marrow where it becomes difficult to treat. PCa cells in the bone marrow may survive, dormant and undetected for many years before patients eventually relapse with metastatic disease. Bone marrow is a complex tissue that initially is hostile to the PCa cells, Understanding how cancer cells survive in the bone marrow and what changes to the bone microenvironment permit them to switch to an actively growing state could offer new therapeutic strategies to combat metastatic PCa. In this study, we describe a method to culture PCa cells with two other cell types from the bone marrow, stromal cells and endothelial cells, as a way to study the interactions among these cell types. We found that factors produced by bone marrow endothelial cells, but not endothelial cells from other tissues, trigger PCa cells to either die or enter a dormant state, similar to what has been observed in patients when PCa cells initially colonize the bone marrow. Further analysis of the cell interactions within the culture model described in this study will offer increased understanding of PCa interaction with the bone marrow environment.The bone marrow tumor microenvironment (BMTE) is a complex network of cells, extracellular matrix, and sequestered signaling factors that initially act as a hostile environment for disseminating tumor cells (DTCs) from the cancerous prostate. Three-dimensional (3D) culture systems offer an opportunity to better model these complex interactions in reactive stroma, providing contextual behaviors for cancer cells, stromal cells, and endothelial cells. Using a new system designed for the triculture of osteoblastic prostate cancer (PCa) cells, stromal cells, and microvascular endothelial cells, we uncovered a context-specific pro-apoptotic effect of endothelial cells of the bone marrow different from those derived from the lung or dermis. The paracrine nature of this effect was demonstrated by observations that conditioned medium from bone marrow endothelial cells, but not from dermal or lung endothelial cells, led to PCa cell death in microtumors grown in 3D BMTE-simulating hydrogels. Analysis of the phosphoproteome by reverse phase protein analysis (RPPA) of PCa cells treated with conditioned media from different endothelial cells identified the differential regulation of pathways involved in proliferation, cell cycle regulation, and apoptosis. The findings from the RPPA were validated by western blotting for representative signaling factors identified, including forkhead box M1 (FOXM1; proliferation factor), pRb (cell cycle regulator), and Smac/DIABLO (pro-apoptosis) among treatment conditions. The 3D model presented here thus presents an accurate model to study the influence of the reactive BMTE, including stromal and endothelial cells, on the adaptive behaviors of cancer cells modeling DTCs at sites of bone metastasis. These findings in 3D culture systems can lead to a better understanding of the real-time interactions among cells present in reactive stroma than is possible using animal models.

Bleomycin restricts the glycolysis of lymphatic endothelial cells by inhibiting dimeric PKM2 formation: A novel mechanism for lymphatic malformation treatment

Glycolysis is activated in lymphatic endothelial cells and contributes to the development of lymphatic malformations (LMs). Bleomycin (BLM) is the most wildly used sclerosant for LMs, but its mechanisms are unclear.Here, our data showed that BLM suppressed the glycolysis of human dermal lymphatic endothelial cells (HDLECs) via inhibiting the expression and nucleus translocation of pyruvate kinase M2 isoform (PKM2) and inhibited dimeric PKM2 formation. Furthermore, the proliferation of LM lesions was inhibited by BLM through the down-regulation of nuclear PKM2 in the rat model. Additionally, PKM2, especially the nuclear PKM2 along with Ki-67, was inhibited in the lymphatic vessels of BLM-treated LMs. Our findings provide a new molecular mechanism of BLM in LM sclerotherapy treatment.

Lymphoedema conditions disrupt endothelial barrier function in vitro.

Lymphatic vessel contractions generate net antegrade pulsatile lymph flow. By contrast, impaired lymphatic vessels are often associated with lymphoedema and altered lymph flow. The effect of lymphoedema on the lymph flow field and endothelium is not completely known. Here, we characterized the lymphatic flow field of a platelet-specific receptor C-type lectin-like receptor 2 (CLEC2) deficient lymphoedema mouse model. In regions of lymphoedema, collecting vessels were significantly distended, vessel contractility was greatly diminished and pulsatile lymph flow was replaced by quasi-steady flow. In vitro exposure of human dermal lymphatic endothelial cells (LECs) to lymphoedema-like quasi-steady flow conditions increased intercellular gap formation and permeability in comparison to normal pulsatile lymph flow. In the absence of flow, LECs exposed to steady pressure (SP) increased intercellular gap formation in contrast with pulsatile pressure (PP). The absence of pulsatility in steady fluid flow and SP conditions without flow-induced upregulation of myosin light chain (MLCs) regulatory subunits 9 and 12B mRNA expression and phosphorylation of MLCs, in contrast with pulsatile flow and PP without flow. These studies reveal that the loss of pulsatility, which can occur with lymphoedema, causes LEC contraction and an increase in intercellular gap formation mediated by MLC phosphorylation.

LncRNA CASC9 activated by STAT3 promotes the invasion of breast cancer and the formation of lymphatic vessels by enhancing H3K27ac-activated SOX4.

Numerous long noncoding RNAs (lncRNAs) are abnormally expressed in breast cancer (BC), but the underlying mechanisms remain large unknown. Here, we aimed to investigate the functions and mechanisms of lncRNA cancer susceptibility candidate 9 (CASC9) in BC. Western blotting and quantitative real-time PCR (qRT-PCR) were performed to assess gene and protein expression, respectively. The proliferative and metastatic abilities of BC cells were tested by cell counting kit-8 and transwell assays, respectively. The formation of lymphatic vessels was detected by tube formation assay. Chromatin immunoprecipitation (ChIP) and dual luciferase reporter assays were performed to verify molecular interactions. CASC9 was found to be highly expressed in BC tissues and cell lines, and ectopic overexpression was positively associated with tumor volume, TNM stage, and lymph node metastasis. In addition, CASC9 silencing significantly inhibited the proliferation and invasion of BC cells, as well as BC-associated invasion and formation of lymphatic vessels of human dermal lymphatic endothelial cells. Mechanical studies demonstrated that CASC9 could be transcriptionally activated by STAT3 and elevate SOX4 expression by enhancing the acetylation of its promoter region. Our results illustrated that STAT3-activated CASC9 served as a tumor-promoting gene involved in promoting BC invasion and BC-associated formation of lymphatic vessels by upregulating SOX4 through altering H3K27ac level. This finding elucidated a new underlying network of CASC9 in the metastasis of BC.

LB1024 Ashwagandha (Withania somnifera) seed plant exosome-like nanovesicles promote in vitro cellular regeneration of human dermal hair follicle, endothelial and fibroblast cells

To overcome inherent drawbacks associated with human-derived platelet-rich plasma (PRP) and exosome therapeutics in the treatment of hair thinning and other dermatological conditions, plant exosome-like nanovesicles were derived from Ashwagandha (Withania somnifera) seeds (Ash-PELNs) to test their regenerative capacity. Prior Ash-PELN studies indicate topical human safety, tolerability and anagen active hair growth prolongation. The regenerative capacity of Ash-PELNs was evaluated in vitro by cellular proliferation, angiogenesis, and growth factor stimulation measures.

Effect of the snake venom component crotamine on lymphatic endothelial cell responses and lymph transport.

The pathology of snake envenomation is closely tied to the severity of edema in the tissue surrounding the area of the bite. Elucidating the mechanisms that promote the development of such severe edema is critical to a better understanding of how to treat this life-threatening injury. We focused on one of the most abundant venom components in North American viper venom, crotamine, and the effects it has on the cells and function of the lymphatic system. We used RT-PCR to identify the location and relative abundance of crotamine's cellular targets (Kvα channels) within the tissues and cells of the lymphatic system. We used calcium flux, nitrate production, and cell morphometry to determine the effects of crotamine on lymphatic endothelial cells. We used tracer transport, node morphometry, and node deposition to determine the effects of crotamine on lymph transport in vivo. We found that genes that encode targets of crotamine are highly present in lymphatic tissues and cells and that there is a differential distribution of those genes that correlates with phasic contractile activity. We found that crotamine potentiates calcium flux in human dermal lymphatic endothelial cells in response to stimulation with histamine and sheer stress (but not alone) and that it alters the production of nitric oxide in response to shear as well as changes the level of F-actin polymerization of those same cells. Crotamine alters lymphatic transport of large molecular weight tracers to local lymph nodes and is deposited within the node mostly in the immediate subcapsular region. This evidence suggests that snake venom components may have an impact on the function of the lymphatic system. This needs to be studied in greater detail as there are numerous venom components that may have effects on aspects of the lymphatic system. This would not only provide basic information on the pathobiology of snakebite but also provide targets for improved therapeutics to treat snakebite.

A mouse infection model and long-term lymphatic endothelium co-culture system to evaluate drugs against adult Brugia malayi.

The development of new drugs targeting adult-stage lymphatic filarial nematodes is hindered by the lack of a robust long-term in vitro culture model. Testing potential direct-acting and anti-Wolbachia therapeutic candidates against adult lymphatic filariae in vitro requires their propagation via chronic infection of gerbils. We evaluated Brugia malayi parasite burden data from male Mongolian gerbils compared with two immune-deficient mouse strains highly susceptible to B. malayi: CB.17 Severe-Combined Immmuno-Deficient (SCID) and interleukin-4 receptor alpha, interleukin-5 double knockout (IL-4Rα-/-IL-5-/-) mice. Adult worms generated in IL-4Rα-/-IL-5-/- mice were tested with different feeder cells (human embryonic kidney cells, human adult dermal lymphatic endothelial cells and human THP-1 monocyte differentiated macrophages) and comparative cell-free conditions to optimise and validate a long-term in vitro culture system. Cultured parasites were compared against those isolated from mice using motility scoring, metabolic viability assay (MTT), ex vivo microfilariae release assay and Wolbachia content by qPCR. A selected culture system was validated as a drug screen using reference anti-Wolbachia (doxycycline, ABBV-4083 / flubentylosin) or direct-acting compounds (flubendazole, suramin). BALB/c IL-4Rα-/-IL-5-/- or CB.17 SCID mice were superior to Mongolian gerbils in generating adult worms and supporting in vivo persistence for periods of up to 52 weeks. Adult females retrieved from BALB/c IL-4Rα-/-IL-5-/- mice could be cultured for up to 21 days in the presence of a lymphatic endothelial cell co-culture system with comparable motility, metabolic activity and Wolbachia titres to those maintained in vivo. Drug studies confirmed significant Wolbachia depletions or direct macrofilaricidal activities could be discerned when female B. malayi were cultured for 14 days. We therefore demonstrate a novel methodology to generate adult B. malayi in vivo and accurately evaluate drug efficacy ex vivo which may be adopted for drug screening with the dual benefit of reducing overall animal use and improving anti-filarial drug development.

Bullous lupus erythematosus with basement membrane deposits of IgD

Bullous lupus erythematosus is an uncommon blistering subepidermal autoimmune disease with characteristic immunopathological nosologic features. A 67-year-old female presented with the sudden appearance of blisters in the upper area of the right chest. Skin biopsies stained with H&amp;E favored the diagnosis of blistering lupus erythematous with a decrease in dermal sebaceous and eccrine glands. Direct immunofluorescence revealed deposits of IgG, complement C3, fibrinogen, IgM, and IgD in the epidermal corneal layer and the basement membrane zone. Reactivity to some dermal endothelial cell junctions and neural receptors was also noted. Also, complement C4 was positive against upper and middle dermal thin fibers. Complement D2 was positive around some enlarged dermal vessels, colocalizing with factor XIIIa. Our case highlights the complex and previously unreported immunologic response features associated with bullous lupus erythematosus.

HMGB1 Promotes Lymphangiogenesis through the Activation of RAGE on M2 Macrophages in Laryngeal Squamous Cell Carcinoma.

Background Receptor for advanced glycation end products (RAGE) is implicated in tumor biology. Released high mobility group box protein 1 (HMGB1) ligand binding to RAGE receptor in tumor cells promotes tumor progression. The mechanisms of HMGB1-RAGE signaling in M2 macrophages involved in lymphangiogenesis in laryngeal carcinoma remain poorly understood. Here, we assessed the effect of HMGB1-RAGE signaling on M2 macrophages in lymphangiogenesis. Methods HMGB1, CD163, and D2-40 in laryngeal squamous cell carcinoma (LSCC, n = 123), laryngeal precursor lesions (LPLs, n = 102), and vocal polyp (VP, n = 55) were analyzed by immunohistochemistry. THP-1 cell-expressed RAGE gene was knocked down and then polarized to M0 macrophages and M2 macrophages. IL-23, TNF-α, TGF-β, and IL-10 were measured by ELISA; IL-1β, IL-12, IL-10, and CCL-13 were evaluated by RT-qPCR, and CD206, CD163, and RAGE were evaluated by western blot to evaluate whether classical M2 macrophages were obtained. Conditioned media from RAGE+/- M0 macrophages and RAGE+/- M2 macrophages incubated in the presence or absence of HMGB1, anti-Toll-like receptor (TLR)2, anti-TLR4 antibodies, and anti-VEGF-C antibodies were collected separately for human dermal lymphatic endothelial cells (HDLEC) for proliferation, migration, lymphangiogenesis assay, and VEGF-C concentration analysis. Results HMGB1 and M2 macrophage densities were increased in LSCC (P < 0.01). HMGB1 and M2 macrophage densities were significantly correlated with lymphatic vessel density (LVD) in LSCC (P < 0.01). The HMGB1 overexpression and higher M2 macrophage density were involved in lymph node metastasis (P < 0.01) and poor prognosis (P < 0.05). In vitro, conditioned medium from HMGB1-stimulated RAGE+ M2 macrophages activated lymphangiogenesis by upregulating the VEGF compared to controls (P < 0.05). On the contrary, RAGE knockdown obviously decreased the corresponding effects of HMGB1-preconditioned M2 macrophages upon HDLEC (P < 0.05). HMGB1-TLR pathway does not significantly increase HDLEC proliferation, migration, and lymphangiogenesis on M2 macrophages. Conclusions HMGB1 promotes lymphangiogenesis by activation of RAGE on M2 macrophages. Targeting RAGE may provide an effective therapeutic strategy against M2 macrophages in LSCC patients with lymph node metastasis.

Lymphangiogenesis contributes to exercise-induced physiological cardiac growth

BackgroundPromoting cardiac lymphangiogenesis exerts beneficial effects for the heart. Exercise can induce physiological cardiac growth with cardiomyocyte hypertrophy and increased proliferation markers in cardiomyocytes. However, it remains unclear whether and how lymphangiogenesis contributes to exercise-induced physiological cardiac growth. We aimed to investigate the role and mechanism of lymphangiogenesis in exercise-induced physiological cardiac growth. MethodsAdult C57BL6/J mice were subjected to 3 weeks of swimming exercise to induce physiological cardiac growth. Oral treatment with vascular endothelial growth factor receptor 3 (VEGFR3) inhibitor SAR131675 was used to investigate whether cardiac lymphangiogenesis was required for exercise-induced physiological cardiac growth by VEGFR3 activation. Furthermore, human dermal lymphatic endothelial cell (LEC)-conditioned medium was collected to culture isolated neonatal rat cardiomyocytes to determine whether and how LECs could influence cardiomyocyte proliferation and hypertrophy. ResultsSwimming exercise induced physiological cardiac growth accompanied by a remarkable increase of cardiac lymphangiogenesis as evidenced by increased density of lymphatic vessel endothelial hyaluronic acid receptor 1–positive lymphatic vessels in the heart and upregulated LYVE-1 and Podoplanin expressions levels. VEGFR3 was upregulated in the exercised heart, while VEGFR3 inhibitor SAR131675 attenuated exercise-induced physiological cardiac growth as evidenced by blunted myocardial hypertrophy and reduced proliferation marker Ki67 in cardiomyocytes, which was correlated with reduced lymphatic vessel density and downregulated LYVE-1 and Podoplanin in the heart upon exercise. Furthermore, LEC-conditioned medium promoted both hypertrophy and proliferation of cardiomyocytes and contained higher levels of insulin-like growth factor-1 and the extracellular protein Reelin, while LEC-conditioned medium from LECs treated with SAR131675 blocked these effects. Functional rescue assays further demonstrated that protein kinase B (AKT) activation, as well as reduced CCAAT enhancer-binding protein beta (C/EBPβ) and increased CBP/p300-interacting transactivators with E (glutamic acid)/D (aspartic acid)–rich-carboxylterminal domain 4 (CITED4), contributed to the promotive effect of LEC-conditioned medium on cardiomyocyte hypertrophy and proliferation. ConclusionOur findings reveal that cardiac lymphangiogenesis is required for exercise-induced physiological cardiac growth by VEGFR3 activation, and they indicate that LEC-conditioned medium promotes both physiological hypertrophy and proliferation of cardiomyocytes through AKT activation and the C/EBPβ-CITED4 axis. These results highlight the essential roles of cardiac lymphangiogenesis in exercise-induced physiological cardiac growth.

Angiopoietin-2-induced lymphatic endothelial cell migration drives lymphangiogenesis via the β1 integrin-RhoA-formin axis.

Lymphangiogenesis is an essential physiological process but also a determining factor in vascular-related pathological conditions. Angiopoietin-2 (Ang2) plays an important role in lymphatic vascular development and function and its upregulation has been reported in several vascular-related diseases, including cancer. Given the established role of the small GTPase RhoA on cytoskeleton-dependent endothelial functions, we investigated the relationship between RhoA and Ang2-induced cellular activities. This study shows that Ang2-driven human dermal lymphatic endothelial cell migration depends on RhoA. We demonstrate that Ang2-induced migration is independent of the Tie receptors, but dependent on β1 integrin-mediated RhoA activation with knockdown, pharmacological approaches, and protein sequencing experiments. Although the key proteins downstream of RhoA, Rho kinase (ROCK) and myosin light chain, were activated, blockade of ROCK did not abrogate the Ang2-driven migratory effect. However, formins, an alternative target of RhoA, were identified as key players, and especially FHOD1. The Ang2-RhoA relationship was explored in vivo, where lymphatic endothelial RhoA deficiency blocked Ang2-induced lymphangiogenesis, highlighting RhoA as an important target for anti-lymphangiogenic treatments.

Cadmium mediates pyroptosis of human dermal lymphatic endothelial cells in a NLRP3 inflammasome-dependent manner.

Pyroptosis is a form of inflammasome-trigged programmed cell death in response to a variety of stimulators, including environmental cytotoxic pollutant Cadmium (Cd). Vascular endothelial cell is one of the first-line cell types of Cd cell toxicity. Studies report that Cd exposure causes pyroptosis in vascular endothelial cells. Vascular and lymphatic endothelial cells have many common properties, but these two cell types are distinguished in gene expression profile and the responsive behaviors to chemokine or physical stimulations. Whether Cd exposure also causes pyroptosis in lymphatic endothelial cells has not been investigated. Here, we found that Cd treatment significantly decreased the viability of human dermal lymphatic endothelial cells (HDLECs). Cd treatment induced inflammasome activation indicated by elevated cleavage of pro-caspase-1 into active form Casp1p20, elevated secretion of pro-inflammatory cytokines and production of reactive oxygen species (ROS). Flow cytometry showed that caspase-1 activity was significantly increased in Cd-treated cells. Moreover, knockdown of NLRP3 effectively rescued Cd-induced inflammasome activation and pyroptosis in HDLECs. Collectively, our results indicated that Cd induced pyroptosis in a NLRP3 inflammasome-dependent manner in lymphatic endothelial cells.

Microphysiological endothelial models to characterize subcutaneous drug absorption.

The high variability in subcutaneous bioavailability of protein therapeutics is poorly understood, contributing to critical delays in patient access to new therapies. Preclinical animal and in vitro models fail to provide a physiologically relevant testbed to parse potential contributors to human bioavailability, therefore new strategies are necessary. Here, we present a microphysiological model of the human hypodermal vasculature at the injection site to study the interactions of administered protein therapeutics within the microenvironment that influence subcutaneous bioavailability. Our model combines human dermal endothelial cells, fibroblasts, and adipocytes, self-assembled into three-dimensional, perfusable microvessels that express relevant extracellular matrix. We demonstrate the utility of the model for measurement of biophysical parameters within the hypodermal microenvironment that putatively impact protein kinetics and distribution at the injection site. We propose that microphysiological models of the subcutaneous space have applications in preclinical development of protein therapeutics intended for subcutaneous administration with optimal bioavailability.