Upregulation Of Angiogenic Factors Research Articles

HIF-1α-dependent upregulation of angiogenic factors by mechanical stimulation in retinal pigment epithelial cells.

Mechanical stimulation as a mimic of drusen formation in the eye increases the expression of angiogenic factors in retinal pigment epithelial (RPE) cells, but the underlying molecular mechanisms remain unclear. We investigated and characterized the effects of mechanical stimulation on the expression of angiogenic factors in RPE cells both in vitro and in a mouse model. Mechanical stimulation increased the expression of vascular endothelial growth factor (VEGF, encoded by VEGFA) and other angiogenesis-related genes in cultured RPE1 cells. The presence of hypoxia-inducible factor 1α (HIF-1α, encoded by HIF1A) was also increased, and both knockdown of HIF-1α and treatment with the HIF-1α inhibitor CAY10585 attenuated the effect of mechanical stimulation on angiogenesis factor gene expression. Signaling by the tyrosine kinase SRC and p38 mitogen-activated protein kinase was involved in HIF-1α activation and consequent angiogenesis-related gene expression induced by mechanical stimulation. Our results suggest that SRC-p38 and HIF-1α signaling are involved in the upregulation of angiogenic factors in RPE cells by mechanical stimulation. Such in vivo suppression of upregulated expression of angiogenesis-related genes by pharmacological inhibitors of HIF-1α suggests a new potential approach to the treatment of age-related macular degeneration.

Tibial cortex transverse transport promotes ischemic diabetic foot ulcer healing via enhanced angiogenesis and inflammation modulation in a novel rat model

BackgroundTibial Cortex Transverse Transport (TTT) represents an innovative surgical method for treating lower extremity diabetic foot ulcers (DFUs), yet its underlying mechanisms remain elusive. Establishing an animal model that closely mirrors clinical scenarios is both critical and novel for elucidating the mechanisms of TTT.MethodsWe established a diabetic rat model with induced hindlimb ischemia to mimic the clinical manifestation of DFUs. TTT was applied using an external fixator for regulated bone movement. Treatment efficacy was evaluated through wound healing assessments, histological analyses, and immunohistochemical techniques to elucidate biological processes.ResultsThe TTT group demonstrated expedited wound healing, improved skin tissue regeneration, and diminished inflammation relative to controls. Marked neovascularization and upregulation of angiogenic factors were observed, with the HIF-1α/SDF-1/CXCR4 pathway and an increase in EPCs being pivotal in these processes. A transition toward anti-inflammatory M2 macrophages indicated TTT's immunomodulatory capacity.ConclusionOur innovative rat model effectively demonstrates the therapeutic potential of TTT in treating DFUs. We identified TTT's roles in promoting angiogenesis and modulating the immune system. This paves the way for further in-depth research and potential clinical applications to improve DFU management strategies.

Probiotic Lactobacillus rhamnosus GG (LGG) restrains the angiogenic potential of colorectal carcinoma cells by activating a proresolving program via formyl peptide receptor 1.

Formyl peptide receptors (FPR1, FPR2 and FPR3) are innate immune sensors of pathogen and commensal bacteria and have a role in colonic mucosa homeostasis. We identified FPR1 as a tumour suppressor in gastric cancer cells due to its ability to sustain an inflammation resolution response with antiangiogenic potential. Here, we investigate whether FPR1 exerts similar functions in colorectal carcinoma (CRC) cells. Since it has been shown that the commensal bacterium Lactobacillus rhamnosus GG (LGG) can promote intestinal epithelial homeostasis through FPR1, we explored the possibility that it could induce proresolving and antiangiogenic effects in CRC cells. We demonstrated that pharmacologic inhibition or genetic deletion of FPR1 in CRC cells caused a reduction of proresolving mediators and a consequent upregulation of angiogenic factors. The activation of FPR1 mediates opposite effects. Proresolving, antiangiogenic and homeostatic functions were also observed upon treatment of CRC cells with supernatant of LGG culture, but not of other lactic acid or nonprobiotic bacteria (i.e. Bifidobacterium bifidum or Escherichia coli). These activities of LGG are dependent on FPR1 expression and on the subsequent MAPK signalling activation. Thus, the innate immune receptor FPR1 could be a regulator of the balance between microbiota, inflammation and cancer in CRC models.

Vasohibin-1 rescues erectile function through up-regulation of angiogenic factors in the diabetic mice

Neovascularization of the erectile tissue emerges as a beneficial curative approach to treat erectile dysfunction (ED). Here we for the first time report the unexpected role of vasohibin-1 (VASH1), mainly known as an anti-angiogenic factor, in restoring erectile function in diabetic mice. A diabetic patient has lower cavernous VASH1 expression than in the potent man. VASH1 was mainly expressed in endothelial cells. There were significant decreases in cavernous endothelial cell and pericyte contents in VASH1 knockout mice compared with those in wild-type mice, which resulted in impairments in erectile function. Intracavernous injection of VASH1 protein successfully restored erectile function in the diabetic mice (~ 90% of control values). VASH1 protein reinstated endothelial cells, pericytes, and endothelial cell–cell junction proteins and induced phosphorylation of eNOS (Ser1177) in the diabetic mice. The induction of angiogenic factors, such as angiopoietin-1 and vascular endothelial growth factor, is responsible for cavernous angiogenesis and the restoration of erectile function mediated by VASH1. Altogether, these findings suggest that VASH1 is proangiogenic in diabetic penis and is a new potential target for diabetic ED.

Attenuation of exaggerated blood pressure responses to exercise: a byproduct of improved vascular function with passive heat therapy?

Attenuation of exaggerated blood pressure responses to exercise: a byproduct of improved vascular function with passive heat therapy?

Effects of Different Vitrification Solutions and Protocol on Follicular Ultrastructure and Revascularization of Autografted Mouse Ovarian Tissue.

Objective Many attempts have been made to preserve fertility by improving the cryopreservation of the ovarian tissue. This current studyaimed to improve of direct cover vitrification (DCV) protocol on follicular preservation and angiogenesis in autografted ovarian tissue. Materials and Methods In this experimental study, sixty five female Balb/c mice (5-6 week-old) were anesthetized and their ovaries were dissected. The left ovaries were vitrified by DCV solution, thawed by descending concentrations of sucrose, and then autografted subcutaneously. The right ovaries were autografted with no vitrification procedure prior to transplantation. The animals were sacrificed under anesthesia on the 7thday after transplantation to obtain ovarian tissue. Follicular quality was assessed by histological and ultrastructure observations, and angiogenesis was examined by immunohistochemical staining and real-time polymerase chain reaction (PCR) analysis. Results The histological and ultrastructure features of the follicles preserved well after vitrification of the ovarian tissue by 10% ethylene glycol (EG) and 10% dimethyl sulfoxide (DMSO). Revascularizationwas manifested prominently in the DCV1-vitrified/grafted ovaries by von Willebrand factor (vWF) and alpha smooth muscle actin (α-SMA) immunostaining. The ovarian tissue vitrified in DCV1 protocol had higher expression levels of angiopoietin-2 (Ang-2) and vascular endothelial growth factor (VEGF) 7 days after autotransplantation (P<0.01). ConclusionThese findings suggest that DCV with 10% of both EG and DMSO, is an effective cryopreservation solution for preservation of good quality follicles as well an upregulation of angiogenic factors after ovarian tissue transplantation.

Upregulation of Angiogenic Factors via Protein Kinase C and Hypoxia-induced Factor-1α Pathways under High-glucose Conditions in the Placenta.

Abnormal glucose metabolism during pregnancy is an established risk factor for preeclampsia (PE). Disruption of the balance between placental angiogenic factors is linked to PE pathophysiology. We examined whether hypoxia-induced factor-1α (HIF-1α) and protein kinase Cβ (PKCβ) are involved in the regulation of placental angiogenic factors under high-glucose conditions in vitro. The human choriocarcinoma cell lines BeWo and JEG-3, and the human trophoblast cell line HTR-8/SVneo were cultured with 10 and 25 mmol/L glucose [control glucose group (CG) and high-glucose group (HG), respectively]. We examined the changes in HIF-1α, soluble fms-like tyrosine kinase-1 (sFlt-1), placental growth factor (PlGF), and vascular endothelial growth factor (VEGF) expression in the CG and HG by real-time PCR and ELISA. PKC activation was also measured by ELISA. The expressions of HIF-1α, sFlt-1, PlGF, and VEGF were significantly higher in the HG than in the CG. PKC activity was significantly increased in the HG. High glucose affected the expression of angiogenic factors in choriocarcinoma cells via the PKCβ and HIF-1α pathways, suggesting their involvement in PE pathogenesis.

3D Protein-Based Bilayer Artificial Skin for the Guided Scarless Healing of Third-Degree Burn Wounds in Vivo.

Severe burn injuries can lead to delays in healing and devastating scar formation. Attempts have been made to develop a suitable skin substitute for the scarless healing of such skin wounds. Currently, there is no effective strategy for completely scarless healing after the thermal injuries. In our recent work, we fabricated and evaluated a 3D protein-based artificial skin made from decellularized human amniotic membrane (AM) and electrospun nanofibrous silk fibroin (ESF) in vitro. We also characterized both biophysical and cell culture investigation to establish in vitro performance of the developed bilayer scaffolds. In this report, we evaluate the appropriate utility of this fabricated bilayered artificial skin in vivo with particular emphasis on healing and scar formation due to the biochemical and biomechanical complexity of the skin. For this work, AM and AM/ESF membranes alone or seeded with adipose-tissue-derived mesenchymal stem cells (AT-MSCs) are implanted on full-thickness burn wounds in mice. The healing efficacy and scar formation are evaluated at 7, 14, and 28 days post-implantation in vivo. Our data reveal that ESF accelerates the wound-healing process through the early recruitment of inflammatory cells such as macrophages into the defective site as well as the up-regulation of angiogenic factors from the AT-MSCs and the facilitation of the remodeling phase. In vivo application of the prepared AM/ESF membrane seeded with the AT-MSCs reduces significantly the post-burn scars. The in vivo data suggest that the potential applications of the AM/ESF bilayered artificial skin may be considered a clinical translational product with stem cells to guide the scarless healing of severe burn injuries.

Abstract A132: Deciphering the molecular regulators of tumor resistance to VEGF-receptor tyrosine kinase inhibitors via in vivo genome-wide RNAi screen

Abstract Inhibiting angiogenesis, which is a shared characteristic of all solid tumors, is a promising approach for treating cancer. However, emerging resistance after initial response is still a drawback. We sought to decipher the molecular landscape of tumor resistance to continuous treatment with VEGF-receptor tyrosine kinase inhibitor (RTKi) sunitinib (SU11248, SU). To address that, we performed serial transplantation of a syngeneic LLC tumor model in C57bl6 mice using a genome-wide lentiviral shRNA library targeting about 39,000 transcripts (150K complexity). The abundance of each single knockdown (kd) was longitudinally traced via molecular barcodes towards development of resistant tumors. After three in vivo panning rounds, distinct shRNA constructs were found to be significantly enriched in tumors passaged under SU selection pressure (p3-SU) compared to those passaged without treatment (p3-ctrl). We focused on validation of three candidate shRNA targeting Kdm5c, Csf1, and Cd44. Knockdown of each of these three genes confirmed their role in emerging resistance to sunitinib in vivo in syngeneic model. Furthermore, knockdown of Kdm5c promoted resistance in 786-0 human renal cell carcinoma xenograft model, in which sunitinib is used as the first-line therapy. Transcriptome analysis of shKdm5c, shCsf1, and shCd44 harboring vs. shCtrl cells suggested that the development of resistance is partly due to modulation of cellular pathways as well as alteration of interaction between cells and their microenvironment. In addition, shKdm5c tumors demonstrated resistance towards pazopanib, an VEGF-RTKi with different inhibitory spectrum compared to sunitinib, which suggests a general mechanism of resistance to inhibitors of VEGF signaling after Kdm5c knockdown. Furthermore, cells with shKdm5c showed an increase in survival under hypoxia induced by VEGF-RTKi. Expression profiling of these cells in hypoxia illustrated a network of genes under Kdm5c control, including three components of PRC2 epigenetic modulator, Ezh2, Eed, and Suz12. We found that knockdown of Kdm5c interrupts the imposed break on these genes under hypoxia. In conclusion, we have shown that loss of Kdm5c, CSf1, and Cd44 renders tumor cells resistant to VEGF-RTKi partly through compensatory upregulation of angiogenic factors. In addition, loss of Kdm5c leads to aberrant activation of a gene-regulatory circuitry controlling cellular response to hypoxia. Citation Format: Azadeh Fahim Golestaneh, Maoyun Sun, Christian Schwager, Zili Tang, Lili Ma, Philip Hahnfeldt, Mahmoud Moustafa, Wilko Weichert, Juergen Debus, Lynn Hlatky, Amir Abdollahi. Deciphering the molecular regulators of tumor resistance to VEGF-receptor tyrosine kinase inhibitors via in vivo genome-wide RNAi screen [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A132.

Tumour-associated glial host cells display a stem-like phenotype with a distinct gene expression profile and promote growth of GBM xenografts

BackgroundLittle is known about the role of glial host cells in brain tumours. However, supporting stromal cells have been shown to foster tumour growth in other cancers.MethodsWe isolated stromal cells from patient-derived glioblastoma (GBM) xenografts established in GFP-NOD/scid mice. With simultaneous removal of CD11b+ immune and CD31+ endothelial cells by fluorescence activated cell sorting (FACS), we obtained a population of tumour-associated glial cells, TAGs, expressing markers of terminally differentiaed glial cell types or glial progenitors. This cell population was subsequently characterised using gene expression analyses and immunocytochemistry. Furthermore, sphere formation was assessed in vitro and their glioma growth-promoting ability was examined in vivo. Finally, the expression of TAG related markers was validated in human GBMs.ResultsTAGs were highly enriched for the expression of glial cell proteins including GFAP and myelin basic protein (MBP), and immature markers such as Nestin and O4. A fraction of TAGs displayed sphere formation in stem cell medium. Moreover, TAGs promoted brain tumour growth in vivo when co-implanted with glioma cells, compared to implanting only glioma cells, or glioma cells and unconditioned glial cells from mice without tumours. Genome-wide microarray analysis of TAGs showed an expression profile distinct from glial cells from healthy mice brains. Notably, TAGs upregulated genes associated with immature cell types and self-renewal, including Pou3f2 and Sox2. In addition, TAGs from highly angiogenic tumours showed upregulation of angiogenic factors, including Vegf and Angiopoietin 2. Immunohistochemistry of three GBMs, two patient biopsies and one GBM xenograft, confirmed that the expression of these genes was mainly confined to TAGs in the tumour bed. Furthermore, their expression profiles displayed a significant overlap with gene clusters defining prognostic subclasses of human GBMs.ConclusionsOur data demonstrate that glial host cells in brain tumours are functionally distinct from glial cells of healthy mice brains. Furthermore, TAGs display a gene expression profile with enrichment for genes related to stem cells, immature cell types and developmental processes. Future studies are needed to delineate the biological mechanisms regulating the brain tumour-host interplay.

In Situ Pluripotency Factor Expression Promotes Functional Recovery From Cerebral Ischemia.

Recovery from ischemic tissue injury can be promoted by cell proliferation and neovascularization. Transient expression of four pluripotency factors (Pou5f1, Sox2, Myc, and Klf4) has been used to convert cell types but never been tested as a means to promote functional recovery from ischemic injury. Here we aimed to determine whether transient in situ pluripotency factor expression can improve neurobehavioral function. Cerebral ischemia was induced by transient bilateral common carotid artery occlusion, after which the four pluripotency factors were expressed through either doxycycline administration into the lateral ventricle in transgenic mice in which the four factors are expressed in a doxycycline-inducible manner. Histologic evaluation showed that this transient expression induced the proliferative generation of astrocytes and/or neural progenitors, but not neurons or glial scar, and increased neovascularization with upregulation of angiogenic factors. Furthermore, in vivo pluripotency factor expression caused neuroprotective effects such as increased numbers of mature neurons and levels of synaptic markers in the striatum. Dysplasia or tumor development was not observed. Importantly, neurobehavioral evaluations such as rotarod and ladder walking tests showed that the expression of the four factors dramatically promoted functional restoration from ischemic injury. These results provide a basis for novel therapeutic modality development for cerebral ischemia.

Abstract 196: Cardiotrophin-1 Promotes Physiologic Cardiac Hypertrophy

The post-natal heart retains a remarkable adaptive capacity, which matches growth of the myocardium to increased physical demands. The beneficial form of myocardial growth is referred to as physiologic hypertrophy, and derives from a compensatory response to conditions that require increased blood volumes such as pregnancy and chronic exercise training. Despite the clear benefits associated with this form of adaptation, the molecular mechanism(s) that initiate or sustain such cardiac remodeling remain obscure. As such, identification of factors that promote physiologic hypertrophy will be of considerable interest to both cardiac biologists and clinicians. Here, we have begun to explore the role of the cytokine, cardiotrophin 1 (CT-1) as a central mediator of physiologic hypertrophy. We demonstrate that primary cardiomyocytes treated with recombinant human CT-1 protein engage a physiologic growth response, adding sarcomeres in series, a cellular/molecular change that is fully reversible with the removal of CT-1. This physiologic hypertrophy response to CT-1 is dominant, as CT-1 administration overrides the pathologic hypertrophy associated with agonists such as phenylephrine. In vivo delivery of human CT-1 protein leads to a rapid (14 days) alteration in the structure of the rat heart, with volume and wall dimension increases characteristic of exercise adaptation, changes that are fully reversible upon cessation of CT-1 treatment. In addition, CT-1 administration mitigates right heart failure in a model of pulmonary arterial hypertension, and sustains cardiac function in the post infarct rat heart. Finally, gene expression analysis revealed that CT-1 treatment leads to up-regulation of angiogenic factors within cardiomyocytes, suggesting that this cytokine engages a paracrine signaling mechanism to ensure that myocardial growth is matched by a robust angiogenesis.

Hypoxia-inducible factor-1 stimulates postnatal lung development but does not prevent O2-induced alveolar injury.

This study investigated whether hypoxia-inducible factor (HIF)-1 influences postnatal vascularization and alveologenesis in mice and whether stable (constitutive-active) HIF could prevent hyperoxia-induced lung injury. We assessed postnatal vessel and alveolar formation in transgenic mice, expressing a stable, constitutive-active, HIF1α-subunit (HIF-1αΔODD) in the distal lung epithelium. In addition, we compared lung function, histology, and morphometry of neonatal transgenic and wild-type mice subjected to hyperoxia. We found that postnatal lungs of HIF-1αΔODD mice had a greater peripheral vessel density and displayed advanced alveolarization compared with control lungs. Stable HIF-1α expression was associated with increased postnatal expression of angiogenic factors, including vascular endothelial growth factor, angiopoietins 1 and 2, Tie2, and Ephrin B2 and B4. Hyperoxia-exposed neonatal HIF-1αΔODD mice exhibited worse lung function but had similar histological and surfactant abnormalities compared with hyperoxia-exposed wild-type controls. In conclusion, expression of constitutive-active HIF-1α in the lung epithelium was associated with increased postnatal vessel growth via up-regulation of angiogenic factors. The increase in postnatal vasculature was accompanied by enhanced alveolar formation. However, stable HIF-1α expression in the distal lung did not prevent hyperoxia-induced lung injury in neonates but instead worsened lung function.

Review of past and present research on experimental models of moyamoya disease

Moyamoya disease (MMD) is characterized by a progressive steno-occlusive disease affecting the terminal portions of the cerebral internal carotid artery (ICA) and by formation of an abnormal vascular network at the base of the brain. Several pathogeneses, including inflammation, immune complex, upregulation of angiogenic factors, and abnormality of endothelial progenitor cells (EPCs) have been hypothesized. However, the mechanisms of MMD are largely unknown, and in vivo and in vitro models of MMD have not yet been established. Previously, inflammation- and immune-complex-related animal models have been reported but failed to reproduce severe stenotic lesions in the terminal portion of ICA. Thereafter, several clinical studies revealed that angiogenic activity of circulating EPCs was defective in MMD patients. These results suggested that the function and quantity of EPCs could be useful as a cellular model of MMD. Very recently, RING finger protein 213 (RNF213) was identified as an MMD susceptibility gene, a discovery that led to the efforts to generate gene mutation-based animal models. Although RNF213 knockout animal models have not yet successfully represented the phenotype of MMD, they have provided new insights into the role of RNF213 in remodeling after vascular injury and postischemic angiogenesis. Furthermore, the use of induced pluripotent stem cells (iPSCs) and an appropriate differentiation protocol have made it possible to obtain abundant quantities of MMD-specific vascular cells. In summary, studies have shown that endothelial cells derived from MMD-iPSCs have impaired angiogenic activity, which is a finding consistent with the results of EPC studies. Further studies are needed to create true MMD-specific experimental models to promote understanding of MMC pathogenesis and aid drug development.

Biology of cerebral arteriovenous malformations with a focus on inflammation.

Cerebral arteriovenous malformations (AVMs) entail a significant risk of intracerebral hemorrhage owing to the direct shunting of arterial blood into the venous vasculature without the dissipation of the arterial blood pressure. The mechanisms involved in the growth, progression and rupture of AVMs are not clearly understood, but a number of studies point to inflammation as a major contributor to their pathogenesis. The upregulation of proinflammatory cytokines induces the overexpression of cell adhesion molecules in AVM endothelial cells, resulting in enhanced recruitment of leukocytes. The increased leukocyte-derived release of metalloproteinase-9 is known to damage AVM walls and lead to rupture. Inflammation is also involved in altering the AVM angioarchitecture via the upregulation of angiogenic factors that affect endothelial cell proliferation, migration and apoptosis. The effects of inflammation on AVM pathogenesis are potentiated by certain single-nucleotide polymorphisms in the genes of proinflammatory cytokines, increasing their protein levels in the AVM tissue. Furthermore, studies on metalloproteinase-9 inhibitors and on the involvement of Notch signaling in AVMs provide promising data for a potential basis for pharmacological treatment of AVMs. Potential therapeutic targets and areas requiring further investigation are highlighted.

CD26/DPPIV down-regulation in endometrial stromal cell migration in endometriosis

To test the hypothesis that endometrial stromal cells (ESCs) in endometriosis exhibit increased cell motility under hypoxia. Prospective case-control study. University research laboratory. Women with endometriosis (n = 18) or benign gynecological disease (n=19). Eutopic ESCs were cultured under normoxia (20% O2) or hypoxia (6.5% O2), and migration and invasion capacity assayed, with pathway-focused polymerase chain reaction (PCR) array and ELISAs performed. CD26/dipeptidyl peptidase IV (DPPIV) expression was determined by flow cytometric analysis and enzymatic activity assay. The ESCs supplemented with Diprotin A (CD26 inhibitor), stromal cell-derived factor-1α, or AMD3100 (C-X-C motif receptor 4; CXCR4 blocker) were assayed for their migratory potential. Endometrial stromal cell migration and invasion under hypoxia. Endometriotic ESCs showed significantly higher migration and invasion through collagen gels under hypoxia compared with nonendometriotic ESCs. The PCR array revealed down-regulation of the migration inhibitor CD26/DPPIV and up-regulation of angiogenic factors (vascular endothelial growth factor A, C-X-C motif Ligand 6; CXCL6) in endometriotic ESCs under hypoxia. The CD26/DPPIV surface expression and activity as well as angiogenic protein secretions suggested that the molecular mechanisms underlying aberrant migratory and angiogenic behavior in endometriotic ESCs. A combinatorial treatment with diprotin A and stromal cell-derived factor-1α effectively enhanced migration and invasion preferentially in endometriotic ESCs cultured hypoxically. Loss of CD26/DPPIV under hypoxia and the subsequent increase in migratory and angiogenic factors may favor conditions for lesion development in endometriosis.

Peroxynitrite Upregulates Angiogenic Factors VEGF-A, BFGF, and HIF-1α in Human Corneal Limbal Epithelial Cells

Corneal neovascularization (NV) is a sight-threatening condition often associated with infection, inflammation, prolonged contact lens use, corneal burns, and acute corneal graft rejection. Macrophages recruited to the cornea release nitric oxide (NO) and superoxide anion (O2(-)), which react together to form the highly toxic molecule peroxynitrite (ONOO(-)). The role of ONOO(-) in upregulating multiple angiogenic factors in cultured human corneal limbal epithelial (HCLE) cells was investigated. Human corneal limbal epithelial cells were incubated with 500 μM of ONOO(-) donor for various times. VEGF-A, BFGF, and hypoxic-inducible factor-alpha (HIF-1α) were investigated via Western blot and RT-PCR was performed for VEGF. Functional assays using human umbilical vein endothelial cells (HUVEC) used conditioned media from ONOO(-)-exposed HCLE cells. Secreted VEGF from conditioned media was detected and analyzed using ELISA. Increased angiogenic factors were observed as early as 4 hours after HCLE exposure to ONOO(-). HIF-1 expression was seen at 4, 6, and 8 hours post-ONOO(-) exposure (P < 0.05). BFGF expression was elevated at 4 hours and peaked at 8 hours after treatment with ONOO(-) (P < 0.005). Increased VEGF-A gene expression was observed at 6 and 8 hours post-ONOO(-) treatment. Functional assays using conditioned media showed increased HUVEC migration and tube formation. Exposure to elevated extracellular concentrations of ONOO(-) results in upregulation of angiogenic factors in HCLE cells. It is possible that, in the setting of inflammation or infection, that exposure to ONOO(-) could be one contributor to the complex initiators of corneal NV. Validation in vivo would identify an additional potential control point for corneal NV.

Wound Healing Improvement with PHD-2 Silenced Fibroblasts in Diabetic Mice

BackgroundHypoxia-inducible factor 1α is the central regulator of the hypoxia-induced response which results in the up-regulation of angiogenic factors. Its activity is under precise regulation of prolyl-hydroxylase domain 2. We hypothesized that PHD2 silenced fibroblasts would increase the expression of angiogenic factors, which might contribute to the improvement of the diabetic wound healing. Materials and Methods50 dB/db mice were employed and randomly assigned into five groups with 10 mice in each: group 1 (untreated cell), group 2 (PHD2 silenced cell), group 3 (L-mimosine treated cells), group 4 (nontargeting siRNA treated cells) and group 5 (sham control). Fibroblasts were cultivated from the dermis of mice in each group and treated with PHD2 targeting siRNA, L-mimosine and non-targeting siRNA respectively. A fraction of the fibroblasts were employed to verify the silencing rate of PHD2 after 48 hours. The autologous fibroblasts (treated and untreated) labeled with adenovirus-GFP were implanted around the wound (Φ6mm), which was created on the dorsum of each mouse. The status of wounds was recorded periodically. Ten days postoperatively, 3 mice from each group were sacrificed and wound tissues were harvested. Molecular biological examinations were performed to evaluate the expressions of cytokines. 28 days postoperatively, the remaining mice were sacrificed. Histological examinations were performed to evaluate the densities of GFP+ cells and capillaries. ResultsThe expression of PHD2 reduced to 12.5%, and the expressions of HIF-1α and VEGFa increased significantly after PHD2 siRNA treatment. With the increasing expressions of HIF-1α and VEGFa, the time to wound closure in group 2 was less than 2 weeks. Increased numbers of GFP+ cells and capillaries were observed in group 2. ConclusionPHD2 siRNA treatment not only increased the expression of HIF1α and VEGFa, but also improved the fibroblast proliferation. These effects might contribute to the improvement of the diabetic wound healing.

Fluvastatin-induced reduction of oxidative stress ameliorates diabetic cardiomyopathy in association with improving coronary microvasculature

Diabetic cardiomyopathy is associated with increased oxidative stress and vascular endothelial dysfunction, which lead to coronary microangiopathy. We tested whether statin-induced redox imbalance improvements could ameliorate diabetic cardiomyopathy and improve coronary microvasculature in streptozotocin-induced diabetes mellitus (DM). Fluvastatin (10 mg/kg/day) or vehicle was orally administered for 12 weeks to rats with or without DM. Myocardial oxidative stress was assessed by NADPH (nicotinamide adenine dinucleotide phosphate) oxidase subunit p22(phox) and gp91(phox) mRNA expression, and myocardial 8-iso-prostaglandin F(2α) (PGF(2α)) levels. Myocardial vascular densities were assessed using anti-CD31 and anti-α-smooth muscle actin (SMA) antibodies. Fluvastatin did not affect blood pressure or plasma cholesterol, but attenuated increased left ventricular (LV) minimum pressure and ameliorated LV systolic dysfunction in DM rats in comparison with vehicle (LV dP/dt, 8.9 ± 1.8 vs 5.4 ± 1.0 × 10(3) mmHg/s, P < 0.05). Myocardial oxidative stress increased in DM, but fluvastatin significantly reduced p22(phox) and gp91(phox) mRNA expression and myocardial PGF(2α) levels. Fluvastatin enhanced myocardial endothelial nitric oxide synthase (eNOS) protein levels and increased eNOS, vascular endothelial growth factor, and hypoxia-inducible factor-1α mRNA expression. CD31-positive cell densities were lower in DM rats than in non-DM rats (28.4 ± 13.2 vs 48.6 ± 4.3/field, P < 0.05) and fluvastatin restored the number (57.8 ± 18.3/field), although there were no significant differences in SMA-positive cell densities between groups. Fluvastatin did not affect cardiac function, oxidative stress, or vessel densities in non-DM rats. These results suggest that beneficial effects of fluvastatin on diabetic cardiomyopathy might result, at least in part, from improving coronary microvasculature through reduction in myocardial oxidative stress and upregulation of angiogenic factor.

Long-term reparative effects of mesenchymal stem cell therapy following neonatal hyperoxia-induced lung injury

Mesenchymal stem cell (MSC) therapy may prevent neonatal hyperoxia-induced lung injury (HILI). There are, however, no clear data on the therapeutic efficacy of MSC therapy in established HILI, the duration of the reparative effects, and the exact mechanisms of repair. The main objective of this study was to evaluate whether the long-term reparative effects of a single intratracheal (IT) dose of MSCs or MSC-conditioned medium (CM) are comparable in established HILI. Newborn rats exposed to normoxia or hyperoxia from postnatal day (P)2)-P16 were randomized to receive IT MSCs, IT CM, or IT placebo (PL) on P9. Alveolarization and angiogenesis were evaluated at P16, P30, and P100. At all time periods, there were marked improvements in alveolar and vascular development in hyperoxic pups treated with MSCs or CM as compared with PL. This was associated with decreased expression of inflammatory mediators and an upregulation of angiogenic factors. Of note, at P100, the improvements were more substantial with MSCs as compared with CM. These data suggest that acute effects of MSC therapy in HILI are mainly paracrine mediated; however, optimum long-term improvement following HILI requires treatment with the MSCs themselves or potentially repetitive administration of CM.