The potassium chloride cotransporters (KCC) family of proteins are widely expressed and are involved in the transepithelial movement of potassium and chloride ions and the regulation of cell volume. KCC activity is high in reticulocytes, and contributes to the dehydration of sickle red blood cells. Because plasma levels of both vascular endothelial growth factor (VEGF) and placental growth factor (PlGF) are elevated in sickle cell individuals, and VEGF has been shown to increase KCC expression in other cells, we hypothesized that VEGF and PlGF influence KCC expression in erythroid cells. Both VEGF and PlGF treatment of human erythroid K562 cells increased both mRNA and protein levels of KCC1, KCC3b, and KCC4. VEGF- and PlGF-mediated cellular signaling involved VEGF-R1 and downstream effectors, specifically, PI-3 kinase, p38 MAP kinase, mTOR, NADPH-oxidase, JNK kinase, and HIF-1α. VEGF and PlGF-mediated transcription of KCC3b and KCC4 involved hypoxia response element (HRE) motifs in their promoters, as demonstrated by promoter analysis, EMSA and ChiP. These results were corroborated in vivo by adenoviral-mediated overexpression of PlGF in normal mice, which led to increased expression of mKCC3 and mKCC4 in erythroid precursors. Our studies show that VEGF and PlGF regulate transcription of KCC3b and KCC4 in erythroid cells via activation of HIF-1α, independent of hypoxia. These studies provide novel therapeutic targets for regulation of cell volume in RBC precursors, and thus, amelioration of dehydration in RBCs in sickle cell disease.

MK2 activation by p38 MAPK selectively induces inflammation in various diseases. We determined if a MK2 inhibitor (MK2i), improves cornea wound healing by inhibiting inflammation caused by burning rat corneas with alkali. Our study, for the first time, demonstrated that MK2i inhibited alkali burn-induced MK2 activation as well as rises in inflammation based on: a) blunting rises in inflammatory index, inflammatory cell infiltration, ED1(+) macrophage and PMN(+) neutrophil infiltration; b) suppressing IL-6 and IL-1β gene expression along with those of macrophage inflammatory protein-1α (MIP-1α), intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1); c) reducing angiogenic gene expression levels and neovascularization (NV) whereas anti-angiogenic PEDF levels increased. In addition, this study found that MK2i did not affect human corneal epithelial cell (HCEC) proliferation and migration and had no detectable side effects on ocular surface integrity. Taken together, MK2i selectively inhibited alkali burn-induced corneal inflammation by blocking MK2 activation, these effects have clinical relevance in the treatment of inflammation related ocular surface diseases.

Tissue expansion techniques play an important role in plastic surgery. How to improve the quality of the expanded skin and shorten the expansion period are still worth investigating. Our previous studies found that a cell-free fat extract (CEFFE) possessed pro-angiogenic and pro-proliferative activities. However, the role of CEFFE on tissue expansion has remained unclear. The purpose of this study was to evaluate the effect of CEFFE on tissue expansion.

The retinopathy of prematurity (ROP), a neovascular retinal disorder presenting in premature infants, is the leading causes of blindness in children. Currently, there is no approved drug therapy for ROP in the U.S., highlighting the urgent unmet clinical need for a novel therapeutic to treat the disease. Secretogranin III (Scg3) was recently identified as a disease-selective angiogenic factor, and Scg3-neutralizing monoclonal antibodies were reported to alleviate pathological retinal neovascularization in mouse models. In this study, we characterized the efficacy and safety of a full-length humanized anti-Scg3 antibody (hAb) to ameliorate retinal pathology in oxygen-induced retinopathy (OIR) mice, a surrogate model of ROP, by implementing histological and functional analyses. Our results demonstrate that the anti-Scg3 hAb outperforms the vascular endothelial growth factor inhibitor aflibercept in terms of efficacy and safety to treat OIR mice. Our findings support the development of anti-Scg3 hAb for clinical application.

Blood vessels in tumors often exhibit abnormal morphology and function, which promotes the growth, metastasis and resistance of tumors to conventional therapies. Therefore, vascular normalization is an emerging strategy to enhance the effectiveness of radiotherapy and chemotherapy when used in combination; however, there is a lack of evidence regarding the optimal schedule for the co-administration of anti-angiogenic and chemotherapeutic drugs. Scheduling treatment is important as the period for normalization is transient, also known as the 'time window'; however, no biomarker has been identified to detect this window. In the present study, recombinant human endostatin (rhES) was employed as an anti-angiogenic agent in xenograft tumor tissue in mice. Following rhES or control (saline) treatment, the density and integrity of tumor vessels were detected by immunofluorescence staining for cluster of differentiation 31 and α-smooth muscle actin; the level of hypoxia in tumor tissue was examined by immunohistochemistry with pimonidazole; the necrotic area was evaluated by hematoxylin and eosin staining; and the level of thrombospondin-1 (TSP-1) in plasma was tested by ELISA. The Cell Counting Kit-8 assay was also used to evaluate the effect of rhES on the proliferation of colon carcinoma SW620 cells. A 'time window' normalized vasculature was determined between day 4 and 6 following rhES treatment, and accompanied by a decrease in hypoxia in tumor tissue. Decreasing plasma TSP-1 levels were consistent with changes in vascular morphology and hypoxia, which exhibited features of normalization. In addition, rhES had no effect on the proliferation of SW620 cells, suggesting that the reduction in TSP-1 was associated with increased oxygen content during vascular normalization, rather than inhibited cell proliferation. In conclusion, TSP-1 may be a potential biomarker for predicting the normalization window of colon cancer vessels.

Hepatocellular carcinoma (HCC) is the fourth leading cause of cancer-associated mortality worldwide. Thus, there is an urgent requirement to identify novel diagnostic and prognostic biomarkers for this disease. The present study aimed to identify the hub genes associated with the progression and prognosis of patients with HCC. A total of three expression profiles of HCC tissues were extracted from the Gene Expression Omnibus (GEO) database, followed by the identification of differentially expressed genes (DEGs) using the GEO2R method. The identified DEGs were assessed for survival significance using Kaplan-Meier analysis. Among the 15 identified DEGs in HCC tissues [cytochrome P450 family 39 subfamily A member 1, cysteine rich angiogenic inducer 61, Fos proto-oncogene, forkhead transcription factor 1 (FOXO1), growth arrest and DNA damage inducible β, Inhibitor of DNA binding 1, interleukin-1 receptor accessory protein, metallothionein-1M, pleckstrin homology-like domain family A member 1, Rho family GTPase 3, serine dehydratase, suppressor of cytokine signaling 2 (SOCS2), tyrosine aminotransferase (TAT), S100 calcium-binding protein P and serine protease inhibitor Kazal-type 1 (SPINK1)]. Low expression levels of FOXO1, SOCS2 and TAT and high SPINK1 expression indicated poor survival outcomes for patients with HCC. In addition, SOCS2 was associated with distinct stages of HCC progression in patients and presented optimal diagnostic value. In vitro functional experiments indicated that overexpression of SOCS2 inhibited HCC cell proliferation and migration. Taken together, the results of the present study suggest that SOCS2 may act as a valuable prognostic marker that is closely associated with HCC progression.

Angiogenesis is a process by which vessels are formed through preexisting ones, and this plays a key role in the progression of solid tumors. However, tumor vessels are influenced by excessive pro-angiogenic factors, resulting in deformed structures that facilitate the intravasation of tumor cells into the circulation and subsequent metastasis. Moreover, abnormal tumor vessels have low blood perfusion and thereby decreased oxygen infusion into tumors. This results in a hostile microenvironment that promotes epithelial-mesenchymal transition (EMT), a process in which epithelial cells lose their polarity and gain increased motility, which is associated with metastasis and invasion. Here, we demonstrate that gold nanoparticles (AuNPs) facilitate tumor vasculature normalization, increase blood perfusion and alleviate hypoxia in melanoma tumors. Additionally, AuNPs were observed to reverse EMT in tumors, accompanied by the alleviation of lung metastasis. These AuNPs inhibited the migration of B16F10 cells and reversed EMT in B16F10 cells, indicating that AuNPs could directly regulate EMT independent of improvements in hypoxia. Taken together, our data demonstrated that AuNPs could induce tumor vasculature normalization and reverse EMT, resulting in decreased melanoma tumor metastasis.

Previous studies have shown that models of depression exhibit structural and functional changes to the neurovascular unit. Thus, we hypothesized that diabetes-related depression might be associated with damage to the hippocampal neurovascular unit. To test this hypothesis, neurons, astrocytes and endothelial cells were isolated from the brain tissues of rat embryos and newborn rats. Hippocampal neurovascular unit co-cultures were produced using the Transwell chamber co-culture system. A model of diabetes-related depression was generated by adding 150 mM glucose and 200 µM corticosterone to the culture system and compared with the neuron + astrocyte and astrocyte + endothelial cell co-culture systems. Western blot assay was used to measure levels of structural proteins in the hippocampal neurovascular unit co-culture system. Levels of basic fibroblast growth factor, angiogenic factor 1, glial cell line-derived neurotrophic factor, transforming growth factor β1, leukemia inhibitory factor and 5-hydroxytryptamine in the hippocampal neurovascular unit co-culture system were measured by enzyme-linked immunosorbent assay. Flow cytometry and terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling staining was used to assess neuronal apoptosis in the hippocampal neurovascular unit. The neurovascular unit triple cell co-culture system had better barrier function and higher levels of structural and secretory proteins than the double cell co-culture systems. In comparison, in the model of diabetes-related depression, the neurovascular unit was damaged with decreased barrier function, poor structural integrity and impaired secretory function. Moreover, neuronal apoptosis was markedly increased, and 5-hydroxytryptamine levels were reduced. These results suggest that diabetes-related depression is associated with structural and functional damage to the neurovascular unit. Our findings provide a foundation for further studies on the pathogenesis of diabetes-related depression.

Since disturbance of angiogenesis predisposes to ischemic injuries, attempts to promote angiogenesis have been made to improve clinical outcomes of patients with many ischemic disorders. While hypoxia inducible factors (HIFs) stimulate vascular remodeling and angiogenesis, hyperlipidemia impairs angiogenesis in response to various pro-angiogenic factors. However, it remains uncertain how HIFs regulate angiogenesis under hyperlipidemia. Here, we report that exposure to low-density lipoprotein (LDL) suppressed in vitro angiogenesis of human brain microvascular endothelial cells. Whereas LDL exposure diminished expression of HIF-1α and HIF-2α induced by hypoxia, it inhibited DMOG- and TNFα-induced HIF-1α and HIF-2α expression in normoxia. Notably, in both hypoxia and normoxia, LDL markedly reduced expression of HIF-1β, a constitutively stable HIF subunit, an event associated with NF-κB inactivation. Moreover, knockdown of HIF-1β down-regulated HIF-1α and HIF-2α expression, in association with increased HIF-1α hydroxylation and 20S proteasome activity after LDL exposure. Significantly, the proteasome inhibitor BSc2118 prevented angiogenesis attenuation by LDL through restoring expression of HIFs. Together, these findings argue that HIF-1β might act as a novel cross-link between the HIF and NF-κB pathways in suppression of angiogenesis by LDL, while proteasome inhibitors might promote angiogenesis by reactivating this signaling cascade under hyperlipidemia.

The majority of long non-coding RNAs (lncRNAs) have been discovered to be overexpressed in pancreatic cancer (PC) and served as promoters in the tumorigenesis of PC, while the inhibitory functions of lncRNAs in the development of PC have not been fully elucidated yet. LncRNA microarray was adopted to analyze the differential expression of lncRNAs in PC tissues and that in normal peritumoral (NP) tissues. Functional role of lncRNA BM466146.1 on PC was evaluated by gain- and loss-of-function experiments in vivo and in vitro. RNA pull-down, RNA immunoprecipitation, luciferase reporter, and Chromatin-immunoprecipitation assays were performed to assess the mechanism of ZNFTR, respectively. The correlation between the expression of ZNFTR and various clinicopathological characteristics was accessed in PC specimens. This study displayed lncRNA BM466146.1 was downregulated in PC tissues and functioned as a suppressor through regulating the expression of adjacent gene Zinc finger protein 24 (ZNF24), which was assigned as ZNFTR. Mechanistically, ZNFTR interacted with activating transcription factor 3 (ATF3) and sequestered ATF3 away from the ZNF24 promoter, which consequently increased the expression of ZNF24. Further, ZNF24 inhibited the proliferative, metastatic, and pro-angiogenic abilities of PC cells by suppressing transcription of vascular endothelial growth factor A (VEGFA). Therefore, the downregulation of ZNFTR in PC led to the decreased expression of ZNF24, which further resulted in the upregulation of VEGFA to facilitate the development of PC. Meanwhile, ZNFTR was transcriptionally inhibited by the HIF-1α/HDAC1 complex-mediated deacetylation. Clinical results further demonstrated that the low expression of ZNFTR was associated with poor overall survival time. Taken together, our results implicated that ZNFTR was a hypoxia-responsive lncRNA, and functioned as an inhibitor by modulating ATF3/ZNF24/VEGFA pathway in PC.

Rationale: Combinations of two or more therapeutic agents targeting different signaling pathways involved in tumor progression can have synergistic anticancer effects. However, combination chemotherapies are greatly limited by the different pharmacokinetics, tumor targeting, and cellular uptake capacities of the combined drugs. We have previously demonstrated the potential synergistic efficacy of paclitaxel (PTX) and the natural anti-angiogenic agent tetramethylpyrazine (TMP) for suppressing ovarian carcinoma growth. An efficient, facile, and smart nanosystem to deliver PTX and TMP simultaneously in vivo is greatly desired. Methods: We constructed a redox-sensitive nanosystem based on the amphiphilic PTX-ss-TMP conjugate, in which PTX and TMP are linked by a disulfide bond. We characterized the structure of the drug conjugate by 1H NMR and LC-MS, and then prepared PTX-ss-TMP NPs by a one-step nanoprecipitation method. We investigated the redox sensitivity, tumor-targeting ability, anticancer efficacy, and anti-angiogenesis activity of PTX-ss-TMP NPs in vitro and in vivo. Results: The amphiphilic PTX-ss-TMP conjugate readily self-assembled into stable nanoparticles in aqueous solution with a low critical association concentration of 1.35 µg/mL, well-defined spherical structure, small particle size (152 nm), high drug loading, redox-responsive drug release, high biocompatibility, and high storage stability. In cancer cells pretreated with GSH-OEt, PTX-ss-TMP NPs exhibited higher cytotoxicity, apoptosis rate, and cell-cycle arrest than monotherapy or combination therapy with free drugs, which was attributed to their improved cellular uptake and rapid intracellular drug release. Additionally, PTX-ss-TMP NPs also had a stronger anti-angiogenesis effect in HUVECs than free drug, which was mediated by VEGFR2-involved downstream signals. Finally, PTX-ss-TMP NPs showed tumor-specific accumulation and excellent antitumor activity in A2780 xenograft mice compared with free drug. Conclusions: These in vitro and in vivo results provide clear evidence that this redox-responsive carrier-free nanosystem with intrinsic amphiphilicity has great potential for combination cancer chemotherapy.