Red Light Mitigates the Deteriorating Placental Extracellular Matrix in Late Onset of Preeclampsia and Improves the Trophoblast Behavior.

Preeclampsia is a serious pregnancy disorder which in extreme cases may lead to maternal and fetal injury or death. Preexisting conditions which increase oxidative stress, e.g., hypertension and diabetes, increase the mother's risk to develop preeclampsia. Previously, we established that when the extracellular matrix is exposed to oxidative stress, trophoblast function is impaired, and this may lead to improper placentation. We investigated how the oxidative ECM present in preeclampsia alters the behavior of first trimester extravillous trophoblasts. We demonstrate elevated levels of advanced glycation end products (AGE) and lipid oxidation end product 4-hydroxynonenal in preeclamptic ECM (28%, and 32% increase vs control, respectively) accompanied with 35% and 82% more 3-chlorotyrosine and 3-nitrotyrosine vs control, respectively. Furthermore, we hypothesized that 670 nm phototherapy, which has antioxidant properties, reverses the observed trophoblast dysfunction as depicted in the improved migration and reduction in apoptosis. Since NO is critical for placentation, we examined eNOS activity in preeclamptic placentas compared to healthy ones and found no differences; however, 670 nm light treatment triggered enhanced NO availability presumably by using alternative NO sources. Light exposure decreased apoptosis and restored trophoblast migration to levels in trophoblasts cultured on preeclamptic ECM. Moreover, 670 nm irradiation restored expression of Transforming Growth Factor (TGFß) and Placental Growth Factor (PLGF) to levels observed in trophoblasts cultured on healthy placental ECM. We conclude the application of 670 nm light can successfully mitigate the damaged placental microenvironment of late onset preeclampsia as depicted by the restored trophoblast behavior.

Abnormal fibrillin-1 expression and chronic oxidative stress mediate endothelial mesenchymal transition in a murine model of systemic sclerosis.

Systemic sclerosis (SSc) is an autoimmune connective tissue disorder characterized by oxidative stress, impaired vascular function, and attenuated angiogenesis. The tight-skin (Tsk(-/+)) mouse is a model of SSc that displays many of the cellular features of the clinical disease. We tested the hypotheses that abnormal fibrillin-1 expression and chronic phospholipid oxidation occur in Tsk(-/+) mice and, furthermore, that these factors precipitate a prooxidant state, collagen-related protein expression, apoptosis, and mesenchymal transition in endothelial cells cultured on Tsk(-/+) extracellular matrix. Human umbilical vein endothelial cells were seeded on microfibrils isolated from skin of C57BL/6J (control) and Tsk(-/+) mice in the presence or absence of chronic pretreatment with the apolipoprotein Apo A-I mimetic D-4F (1 mg·kg(-1)·day(-1) ip for 6 to 8 wk). Nitric oxide-to-superoxide anion ratio was assessed 12 h after culture, and cell proliferation, apoptosis, and phenotype were studied 72 h after culture. Tsk(-/+) mice demonstrated abnormal "big fibrillin" expression (405 kDa) by Western blot analysis compared with control. Endothelial cells cultured on microfibrils prepared from Tsk(-/+) mice demonstrated reduced proliferation, a prooxidant state (reduced nitric oxide-to-superoxide anion ratio), increased apoptosis, and collagen-related protein expression associated with mesenchymal transition. Chronic D-4F pretreatment of Tsk(-/+) mice attenuated many of these adverse effects. The findings demonstrate that abnormal fibrillin-1 expression and chronic oxidative stress mediate endothelial mesenchymal transition in Tsk(-/+) mice. This mesenchymal transition may contribute to the reduction in angiogenesis that is known to occur in this model of SSc.

Non-thermal Infrared Light Treatment of Ischemia/Reperfusion Injury and Subsequent Analysis of Macrophage Differentiation.

Tissue damage and necrosis from inflammatory processes are a consequence of ischemia reperfusion injury (IRI). In skeletal muscle, ischemia reduces the aerobic energy capacity of muscle cells, leading to adverse biochemical alterations and inflammation. The goal of this study is to show that exposure to near-infrared light (NIR) during a period of ischemia reduces IRI by decreasing necrosis and inflammation in addition to decreasing proinflammatory M1 and increasing protective M2 macrophages. C57/Bl6 mice underwent unilateral tourniquet-induced hindlimb ischemia for 3 h followed by reperfusion for either 15 or 30 min. Mice were randomly assigned to 3 groups. Group 1 underwent IRI with 30 min reperfusion. Group 2 underwent IRI with a 15 min reperfusion. Each group consisted of 50% no-NIR and 50% NIR-treated mice with exposure of 50 mW/cm2 for 5 min/1 h after tourniquet closure. Group 3 were sham animals anesthetized for 3 h omitting IRI. Laser doppler flow imaging was performed on all mice to confirm ischemia and reperfusion. Flow data were expressed as the ratio of ischemic limb and the contralateral control. The mice were euthanized after reperfusion, and the quadriceps and gastrocnemius were harvested. Immunoprecipitation and western blot of macrophage-markers CD68 (M1) and CD206 (M2) were performed and normalized to CD14 expression. The expression of the inflammatory markers CXCL1 and CXCL5 was significantly reduced by NIR in the IRI group. A significant decrease in CD68 and an increase in CD206 expression was observed in animals receiving IR and NIR. Tissue necrosis was decreased by NIR in the IRI group, as visualized by 2,3,5-triphenyltetrazolium chloride (TTC) staining. The findings demonstrate that exposure to NIR reduced IRI and improved tissue survival. NIR reduced inflammation, decreased proinflammatory M1, and increased protective M2 macrophages. Exposure to NIR reduced inflammation and enhanced regeneration, leading to tissue protection following ischemia.

Inhibition of myeloperoxidase increases revascularization and improves blood flow in a diabetic mouse model of hindlimb ischaemia.

OBJECTIVE: Diabetes mellitus is a significant risk factor for peripheral artery disease. Diabetes mellitus induces chronic states of oxidative stress and vascular inflammation that increase neutrophil activation and release of myeloperoxidase. The goal of this study is to determine whether inhibiting myeloperoxidase reduces oxidative stress and neutrophil infiltration, increases vascularization, and improves blood flow in a diabetic murine model of hindlimb ischaemia. METHODS: Leptin receptor-deficient (db/db) mice were subjected to hindlimb ischaemia. Ischaemic mice were treated with N-acetyl-lysyltyrosylcysteine-amide (KYC) to inhibit myeloperoxidase. After ligating the femoral artery, effects of treatments were determined with respect to hindlimb blood flow, neutrophil infiltration, oxidative damage, and the capability of hindlimb extracellular matrix to support human endothelial cell proliferation and migration. RESULTS: KYC treatment improved hindlimb blood flow at 7 and 14 days in db/db mice; decreased the formation of advanced glycation end products, 4-hydroxynonenal, and 3-chlorotyrosine; reduced neutrophil infiltration into the hindlimbs; and improved the ability of hindlimb extracellular matrix from db/db mice to support endothelial cell proliferation and migration. CONCLUSION: These results demonstrate that inhibiting myeloperoxidase reduces oxidative stress in ischaemic hindlimbs of db/db mice, which improves blood flow and reduces neutrophil infiltration such that hindlimb extracellular matrix from db/db mice supports endothelial cell proliferation and migration.

Vasodilation of Isolated Vessels and the Isolation of the Extracellular Matrix of Tight-skin Mice.

The interferon regulatory factor 5 (IRF5) is crucial for cells to determine if they respond in a pro-inflammatory or anti-inflammatory fashion. IRF5's ability to switch cells from one pathway to another is highly attractive as a therapeutic target. We designed a decoy peptide IRF5D with a molecular modeling software for designing small molecules and peptides. IRF5D inhibited IRF5, reduced alterations in extracellular matrix, and improved endothelial vasodilation in the tight-skin mouse (Tsk/+). The Kd of IRF5D for recombinant IRF5 is 3.72 ± 0.74 x 10-6 M as determined by binding experiments using biolayer interferometry experiments. Endothelial cells (EC) proliferation and apoptosis were unchanged using increasing concentrations of IRF5D (0 to 100 µg/mL, 24 h). Tsk/+ mice were treated with IRF5D (1 mg/kg/d subcutaneously, 21 d). IRF5 and ICAM expressions were decreased after IRF5D treatment. Endothelial function was improved as assessed by vasodilation of facialis arteries from Tsk/+ mice treated with IRF5D compared to Tsk/+ mice without IRF5D treatment. As a transcription factor, IRF5 traffics from the cytosol to the nucleus. Translocation was assessed by immunohistochemistry on cardiac myocytes cultured on the different cardiac extracellular matrices. IRF5D treatment of the Tsk/+ mouse resulted in a reduced number of IRF5 positive nuclei in comparison to the animals without IRF5D treatment (50 µg/mL, 24 h). These findings demonstrate the important role that IRF5 plays in inflammation and fibrosis in Tsk/+ mice.

An IRF5 Decoy Peptide Reduces Myocardial Inflammation and Fibrosis and Improves Endothelial Cell Function in Tight-Skin Mice.

Interferon regulatory factor 5 (IRF5) has been called a "master switch" for its ability to determine whether cells mount proinflammatory or anti-inflammatory responses. Accordingly, IRF5 should be an attractive target for therapeutic drug development. Here we report on the development of a novel decoy peptide inhibitor of IRF5 that decreases myocardial inflammation and improves vascular endothelial cell (EC) function in tight-skin (Tsk/+) mice. Biolayer interferometry studies showed the Kd of IRF5D for recombinant IRF5 to be 3.72 ± 0.74x10-6M. Increasing concentrations of IRF5D (0-100 µg/mL, 24h) had no significant effect on EC proliferation or apoptosis. Treatment of Tsk/+ mice with IRF5D (1mg/kg/d subcutaneously, 21d) reduced IRF5 and ICAM-1 expression and monocyte/macrophage and neutrophil counts in Tsk/+ hearts compared to expression in hearts from PBS-treated Tsk/+ mice (p<0.05). EC-dependent vasodilatation of facialis arteries isolated from PBS-treated Tsk/+ mice was reduced (~15%). IRF5D treatments (1mg/kg/d, 21d) improved vasodilatation in arteries isolated from Tsk/+ mice nearly 3-fold (~45%, p<0.05), representing nearly 83% of the vasodilatation in arteries isolated from C57Bl/6J mice (~55%). IRF5D (50µg/mL, 24h) reduced nuclear translocation of IRF5 in myocytes cultured on both Tsk/+ cardiac matrix and C57Bl/6J cardiac matrix (p<0.05). These data suggest that IRF5 plays a causal role in inflammation, fibrosis and impaired vascular EC function in Tsk/+ mice and that treatment with IRF5D effectively counters IRF5-dependent mechanisms of inflammation and fibrosis in the myocardium in these mice.

Porous ongrowth surfaces alter osteoblast maturation and mineralization.

Implant fixation through osseointegration is essential for the success of uncemented total joint arthroplasty, and nature and composition of implant surface play a critical role in this process. Despite widespread use of uncemented implants, the extent of bone ingrowth into implants is generally only a small percentage of the total implant surface. An understanding of the processes whereby bone cells grow into and multiply on porous surfaces is critical for the design and manufacture of implants that maximize ingrowth and implant fixation. A wide variety of implant materials are currently utilized for uncemented total joint arthroplasty, including titanium mesh, cobalt chromium beads, and tantalum deposited on a carbon network. Despite differences in physical and chemical properties of these materials, all have functioned well clinically. Therefore, the goals of this study were to compare and contrast the effects of these materials on the proliferation, phenotypic maturation, and mineralization of osteoblasts. Disks of porous tantalum, titanium mesh, and cobalt chromium beaded surfaces were fabricated and processed employing the same methods used to produce implants, including packaging and sterilization. Preosteoblasts were plated on disks, cellular morphology was evaluated by scanning electron microscopy. Osteoblast proliferation was significantly higher on the porous tantalum compared to other implant surfaces. Alkaline phosphatase activity, osteocalcin secretion, and upregulation of RUNX2 were inversely proportional to the rate of proliferation. Mineralization of osteoblasts paralleled the rate of proliferation. These findings suggest that proliferation of osteoblasts into the interstices of implant materials along with delayed maturation were favorable for increased bone ongrowth and ultimately implant stabilization.

Metal ions activate vascular endothelial cells and increase lymphocyte chemotaxis and binding.

Metal on metal articulations in hip arthroplasty offer advantages, including lower volumetric wear compared to conventional metalonpolyethylene bearings, and increased resistance to dislocation. Reports described early failures, with histologic features similar to a Type IV immune response. Mechanisms by which metal wear products cause this reaction are not completely understood. We hypothesized a mechanism through direct activation of endothelial cells (ECs) by metal ions, resulting in both vasculitis and accumulation of lymphocytes without prior immune sensitization. Effects of metal ions were evaluated using human ECs in culture. Alterations in chemotactic proteins IL8 and MCP1 were assessed, as was upregulation of the adhesion molecule ICAM-1 and lymphocyte binding to ECs. Cobalt increased secretion of IL8 and MCP1 significantly, and upregulated the expression of ICAM-1 in ECs compared to stimulation by chromium and controls. Binding of lymphocytes to ECs and transEC migration were both significantly increased by cobalt but not chromium. These findings suggest that cobalt contributes more to the activation of ECs and lymphocyte binding than chromium without an allergic response. Some of the adverse tissue reactions to implants with components made of cobalt-chromium-molybdenium alloys may be due in part to activation of the endothelium by metal ions.

Transient repetitive exposure to low level light therapy enhances collateral blood vessel growth in the ischemic hindlimb of the tight skin mouse.

The tight skin mouse (Tsk(-/+)) is a model of scleroderma characterized by impaired vasoreactivity, increased oxidative stress, attenuated angiogenic response to VEGF and production of the angiogenesis inhibitor angiostatin. Low-level light therapy (LLLT) stimulates angiogenesis in myocardial infarction and chemotherapy-induced mucositis. We hypothesize that repetitive LLLT restores vessel growth in the ischemic hindlimb of Tsk(-/+) mice by attenuating angiostatin and enhancing angiomotin effects in vivo. C57Bl/6J and Tsk(-/+) mice underwent ligation of the femoral artery. Relative blood flow to the foot was measured using a laser Doppler imager. Tsk(-/+) mice received LLLT (670 nm, 50 mW cm(-2), 30 J cm(-2)) for 10 min per day for 14 days. Vascular density was determined using lycopersicom lectin staining. Immunofluorescent labeling, Western blot analysis and immunoprecipitation were used to determine angiostatin and angiomotin expression. Recovery of blood flow to the ischemic limb was reduced in Tsk(-/+) compared with C57Bl/6 mice 2 weeks after surgery. LLLT treatment of Tsk(-/+) mice restored blood flow to levels observed in C57Bl/6 mice. Vascular density was decreased, angiostatin expression was enhanced and angiomotin depressed in the ischemic hindlimb of Tsk(-/+) mice. LLLT treatment reversed these abnormalities. LLLT stimulates angiogenesis by increasing angiomotin and decreasing angiostatin expression in the ischemic hindlimb of Tsk(-/+) mice.