A potential novel therapeutic target in diabetic retinopathy: a chemokine receptor (CCR2/CCR5) inhibitor reduces retinal vascular leakage in an animal model.

PURPOSE: We have previously shown that the chemokine CCL2 plays an important role in monocyte trafficking into the retina and alteration of the BRB in an animal model of diabetic retinopathy. In this study, we examined the effect of pharmacologically targeting the chemokine pathway to reduce the increased retinal vascular permeability in this model. METHODS: C57BL/6 J mice were made diabetic using streptozotocin. After 4 months of diabetes, mice (n = 10) were treated by intraperitoneal injections of TAK-779 (dual CCR2/CCR5 inhibitor) (30 mg/kg) daily for 2 weeks. Retinal vascular permeability and protein expression were done using western blot. The SDF-1 levels were measured by ELISA. Immune cell infiltration in the retinas was measured using flow cytometry. RESULTS: The dual inhibitor significantly decreased retinal vascular permeability in diabetic animals. There was a significant reduction in macrophage/microglia infiltration in the retinas of treated animals. Levels of SDF-1 and ICAM-1 were significantly reduced and the tight junction protein ZO-1 level was increased, and phospho-VE-Cad was significantly reduced with drug treatment. CONCLUSIONS: A chemokine receptor inhibitor (CCR2/CCR5) can reduce retinal vascular permeability in diabetic animals. Targeting the chemokine pathway pharmacologically may be used as a novel therapeutic strategy in management of diabetic macular edema.

Medical Student Attitudes toward USMLE Step 1 and Health Systems Science - A Multi-Institutional Survey.

Phenomenon: Because of its importance in residency selection, the United States Medical Licensing Examination Step 1 occupies a critical position in medical education, stimulating national debate about appropriate score use, equitable selection criteria, and the goals of undergraduate medical education. Yet, student perspectives on these issues and their implications for engagement with health systems science-related curricular content are relatively underexplored. Approach: We conducted an online survey of medical students at 19 American allopathic medical schools from March-July, 2019. Survey items were designed to elicit student opinions on the Step 1 examination and the impact of the examination on their engagement with new, non-test curricular content related to health systems science. Findings: A total of 2856 students participated in the survey, representing 23.5% of those invited. While 87% of students agreed that doing well on the Step 1 exam was their top priority, 56% disagreed that studying for Step 1 had a positive impact on engagement in the medical school curriculum. Eighty-two percent of students disagreed that Step 1 scores should be the top item residency programs use to offer interviews. When asked whether Step 1 results should be reported pass/fail with no numeric score, 55% of students agreed, while 33% disagreed. The majority of medical students agreed that health systems science topics were important but disagreed that studying for Step 1 helped learn this content. Students reported being more motivated to study a topic if it was on the exam, part of a course grade, prioritized by residency program directors, or if it would make them a better physician in the future. Insights: These results confirm the primacy of the United States Medical Licensing Examination Step 1 exam in preclinical medical education and demonstrate the need to balance the objectives of medical licensure and residency selection with the goals of the broader medical profession. The survey responses suggest several potential solutions to increase student engagement in health systems science curricula which may be especially important after Step 1 examination results are reported as pass/fail.

Heme-Dependent ER Stress Apoptosis: A Mechanism for the Selective Toxicity of the Dihydroartemisinin, NSC735847, in Colorectal Cancer Cells.

Colorectal cancer (CRC) is a leading cause of cancer death in the United States. Artemisinin derivatives, including the dihydroartemisinin (DHA) monomers, are widely used as clinical agents for the treatment of malaria. Numerous studies demonstrate that these molecules also display antineoplastic activity with minimal toxicity. Of interest, dimeric DHA molecules are more active than their monomeric counterparts. Our previous data showed that the DHA dimer, NSC735847, was a potent inducer of death in different cancer cell types. However, the mechanism of action and activity of NSC735847 in colon cancer cells was not explored. The present study investigated the anticancer activity of NSC735847 and four structurally similar analog in human tumorigenic (HT-29 and HCT-116) and non-tumorigenic (FHC) colon cell lines. NSC735847 was more cytotoxic toward tumorigenic than non-tumorigenic colonocytes. In addition, NSC735847 exhibited greater cytotoxicity and tumor selectivity than the NSC735847 derivatives. To gain insight into mechanisms of NSC735847 activity, the requirement for endoplasmic reticulum (ER) stress and oxidative stress was tested. The data show that ER stress played a key role in the cytotoxicity of NSC735847 while oxidative stress had little impact on cell fate. In addition, it was observed that the cytotoxic activity of NSC735847 required the presence of heme, but not iron. The activity of NSC735847 was then compared to clinically utilized CRC therapeutics. NSC735847 was cytotoxic toward colon tumor cells at lower concentrations than oxaliplatin (OX). In addition, cell death was achieved at lower concentrations in colon cancer cells that were co-treated with folinic acid (Fol), 5-FU (F), and NSC735847 (FolFNSC), than Fol, F, and OX (FolFOX). The selective activity of NSC735847 and its ability to induce cytotoxicity at low concentrations suggest that NSC735847 may be an alternative for oxaliplatin in the FolFOX regimen for patients who are unable to tolerate its adverse effects.

Transcriptomics analysis of pericytes from retinas of diabetic animals reveals novel genes and molecular pathways relevant to blood-retinal barrier alterations in diabetic retinopathy.

Selective pericyte loss, the histological hallmark of early diabetic retinopathy (DR), enhances the breakdown of the blood-retinal barrier (BRB) in diabetes. However, the role of pericytes on BRB alteration in diabetes and the signaling pathways involved in their effects are currently unknown. To understand the role of diabetes-induced molecular alteration of pericytes, we performed transcriptomic analysis of sorted retinal pericytes from mice model of diabetes. Retinal tissue from non-diabetic and diabetic (duration 3 months) mouse eyes (n = 10 in each group) were used to isolate pericytes through fluorescent activated cell sorting (FACS) using pericyte specific fluorescent antibodies, PDGFRb-APC. For RNA sequencing and qPCR analysis, a cDNA library was generated using template switching oligo and the resulting libraries were sequenced using paired-end Illumina sequencing. Molecular functional pathways were analyzed using differentially expressed genes (DEGs). Differential expression analysis revealed 217 genes significantly upregulated and 495 genes downregulated, in pericytes isolated from diabetic animals. These analyses revealed a core set of differentially expressed genes that could potentially contribute to the pericyte dysfunction in diabetes and highlighted the pattern of functional connectivity between key candidate genes and blood retinal barrier alteration mechanisms. The top up-regulated gene list included: Ext2, B3gat3, Gpc6, Pip5k1c and Pten and down-regulated genes included: Notch3, Xbp1, Gpc4, Atp1a2 and AKT3. Out of these genes, we further validated one of the down regulated genes, Notch 3 and its role in BRB alteration in diabetic retinopathy. We confirmed the downregulation of Notch3 expression in human retinal pericytes exposed to Advanced Glycation End-products (AGEs) treatment mimicking the chronic hyperglycemia effect. Exploration of pericyte-conditioned media demonstrated that loss of NOTCH3 in pericyte led to increased permeability of endothelial cell monolayers. Collectively, we identify a role for NOTCH3 in pericyte dysfunction in diabetes. Further validation of other DEGs to identify cell specific molecular change through whole transcriptomic approach in diabetic retina will provide novel insight into the pathogenesis of DR and novel therapeutic targets.

Do Genomic Factors Play a Role in Diabetic Retinopathy?

Although there is strong clinical evidence that the control of blood glucose, blood pressure, and lipid level can prevent and slow down the progression of diabetic retinopathy (DR) as shown by landmark clinical trials, it has been shown that these factors only account for 10% of the risk for developing this disease. This suggests that other factors, such as genetics, may play a role in the development and progression of DR. Clinical evidence shows that some diabetics, despite the long duration of their diabetes (25 years or more) do not show any sign of DR or show minimal non-proliferative diabetic retinopathy (NPDR). Similarly, not all diabetics develop proliferative diabetic retinopathy (PDR). So far, linkage analysis, candidate gene studies, and genome-wide association studies (GWAS) have not produced any statistically significant results. We recently initiated a genomics study, the Diabetic Retinopathy Genetics (DRGen) Study, to examine the contribution of rare and common variants in the development of different phenotypes of DR, as well as their responsiveness to anti-VEGF treatment in diabetic macular edema (DME). Our preliminary findings reveal a novel set of genetic variants involved in the angiogenesis and inflammatory pathways that contribute to DR progression or protection. Further investigation of variants can help to develop novel biomarkers and lead to new therapeutic targets in DR.

Cathepsin D plays a role in endothelial-pericyte interactions during alteration of the blood-retinal barrier in diabetic retinopathy.

Inflammation plays an important role in the pathogenesis of diabetic retinopathy. We have previously demonstrated the effect of cathepsin D (CD) on the mechanical disruption of retinal endothelial cell junctions and increased vasopermeability, as well as increased levels of CD in retinas of diabetic mice. Here, we have also examined the effect of CD on endothelial-pericyte interactions, as well as the effect of dipeptidyl peptidase-4 (DPP4) inhibitor on CD in endothelial-pericyte interactions in vitro and in vivo. Cocultured cells that were treated with pro-CD demonstrated a significant decrease in the expression of platelet-derived growth factor receptor-ß, a tyrosine kinase receptor that is required for pericyte cell survival; N-cadherin, the key adherens junction protein between endothelium and pericytes; and increases in the vessel destabilizing agent, angiopoietin-2. The effect was reversed in cells that were treated with DPP4 inhibitor along with pro-CD. With pro-CD treatment, there was a significant increase in the phosphorylation of the downstream signaling protein, PKC-α, and Ca2+/calmodulin-dependent protein kinase II in endothelial cells and pericytes, which disrupts adherens junction structure and function, and this was significantly reduced with DPP4 inhibitor treatment. Increased CD levels, vasopermeability, and alteration in junctional-related proteins were observed in the retinas of diabetic rats, which were significantly changed with DPP4 inhibitor treatment. Thus, DPP4 inhibitors may be used as potential adjuvant therapeutic agents to treat increased vascular leakage observed in patients with diabetic macular edema.-Monickaraj, F., McGuire, P., Das, A. Cathepsin D plays a role in endothelial-pericyte interactions during alteration of the blood-retinal barrier in diabetic retinopathy.

Novel therapeutic targets in diabetic macular edema: Beyond VEGF.

The leading cause of major vision loss in diabetic persons is diabetic macular edema (DME). The hallmark feature of diabetic retinopathy is the alteration of the blood-retinal barrier (BRB). Inflammation plays a crucial role in DME with involvement of several chemokines and cytokines including vascular endothelial growth factor (VEGF). VEGF is a potent cytokine and vaso-permeability factor that has been targeted in multiple, large clinical trials. Multiple anti-VEGF drugs are widely used in the treatment of diabetic macular edema (DME) as the first line of treatment, and have been shown to be effective in vision improvement and prevention of vision loss. However, many DME patients do not show complete response to anti-VEGF drugs despite multiple intravitreal injections with these drugs. Also, the effect seems to be transient in those responders, and many patients do not show complete resolution of fluid. This article summarizes the mechanisms other than VEGF, and how these novel factors can be targeted as promising therapies of DME.

Myocutaneous revascularization following graded ischemia in lean and obese mice.

BACKGROUND: Murine models of diabetes and obesity have provided insight into the pathogenesis of impaired epithelialization of excisional skin wounds. However, knowledge of postischemic myocutaneous revascularization in these models is limited. MATERIALS AND METHODS: A myocutaneous flap was created on the dorsum of wild type (C57BL/6), genetically obese and diabetic (ob/ob, db/db), complementary heterozygous (ob+/ob-, db+/db-), and diet-induced obese (DIO) mice (n=48 total; five operative mice per strain and three unoperated mice per strain as controls). Flap perfusion was documented by laser speckle contrast imaging. Local gene expression in control and postoperative flap tissue specimens was determined by quantitative reverse transcription polymerase chain reaction (RT-PCR). Image analysis of immunochemically stained histologic sections confirmed microvascular density and macrophage presence. RESULTS: Day 10 planimetric analysis revealed mean flap surface area necrosis values of 10.8%, 12.9%, 9.9%, 0.4%, 1.4%, and 23.0% for wild type, db+/db-, ob+/ob-, db/db, ob/ob, and DIO flaps, respectively. Over 10 days, laser speckle imaging documented increased perfusion at all time points with revascularization to supranormal perfusion in db/db and ob/ob flaps. In contrast, wild type, heterozygous, and DIO flaps displayed expected graded ischemia with failure of perfusion to return to baseline values. RT-PCR demonstrated statistically significant differences in angiogenic gene expression between lean and obese mice at baseline (unoperated) and at day 10. CONCLUSION: Unexpected increased baseline skin perfusion and augmented myocutaneous revascularization accompanied by a control proangiogenic transcriptional signature in genetically obese mice compared to DIO and lean mice are reported. In future research, laser speckle imaging has been planned to be utilized in order to correlate spatiotemporal wound reperfusion with changes in cell recruitment and gene expression to better understand the differences in wound microvascular biology in lean and obese states.

Novel pharmacotherapies in diabetic retinopathy: Current status and what's in the horizon?

The blood-retinal barrier (BRB) alteration is the hallmark feature of diabetic retinopathy. Vascular endothelial growth factor (VEGF) is a potent vasopermeability factor that has been implicated in the pathogenesis of BRB alteration. Inflammation also plays a crucial role in this process with involvement of several chemokines and cytokines. Multiple anti-VEGF drugs are widely used as in the treatment of diabetic macular edema (DME) as well as proliferative diabetic retinopathy. Several clinical trials have proved the beneficial effects of these drugs in improvement of vision and prevention of vision loss. However, the response to anti-VEGF drugs in DME is not complete in a significant number of patients. The effect seems transient in this latter group, and many patients do not show complete resolution of fluid. Potential novel therapies targeting molecules beyond VEGF are being developed and examined in clinical trials.

Cathepsin D: an Mϕ-derived factor mediating increased endothelial cell permeability with implications for alteration of the blood-retinal barrier in diabetic retinopathy.

Inflammation plays an important role in the pathogenesis of diabetic retinopathy (DR). We have previously reported increased monocyte (Mono) trafficking into the retinas of diabetic animals. In this study, we have examined the effect of activated Monos on retinal endothelial cells (ECs). The U937 Mϕ-conditioned medium (CM) significantly decreased the transendothelial resistance of EC monolayers as measured by electric cell-substrate impedance sensing (P= 0.007). The CM was fractioned, and the effective fraction (30-100 kDa) was analyzed by liquid chromatography-mass spectrometry, and cathepsin D (CD) was identified as a major secreted product. Immunoprecipitated CD resulted in decreased resistance in ECs (P= 0.006). The specificity of CD in mediating alterations of the EC barrier was confirmed using small interfering RNA. The decreased resistance correlated with a significantly increased gap between ECs. CD altered the Ras homolog gene family, member A/Rho-associated kinase pathway with increased stress actin filament formation in the EC layer. Increased CD levels were found in the retinas of diabetic mice (3-fold) and serum samples of patients with diabetic macular edema (1.6-fold) measured by Western blot and ELISA. These findings suggest an important role for Mϕ-derived CD in altering the blood-retinal barrier and reveal a potential therapeutic target in the treatment of DR.-Monickaraj, F., McGuire, P. G., Nitta, C. F., Ghosh, K., Das, A. Cathepsin D: an Mϕ-derived factor mediating increased endothelial cell permeability with implications for alteration of the blood-retinal barrier in diabetic retinopathy.

Basement membrane stiffening promotes retinal endothelial activation associated with diabetes.

Endothelial activation is a hallmark of the high-glucose (HG)-induced retinal inflammation associated with diabetic retinopathy (DR). However, precisely how HG induces retinal endothelial activation is not fully understood. We hypothesized that HG-induced up-regulation of lysyl oxidase (LOX), a collagen-cross-linking enzyme, in retinal capillary endothelial cells (ECs) enhances subendothelial basement membrane (BM) stiffness, which, in turn, promotes retinal EC activation. Diabetic C57BL/6 mice exhibiting a 70 and 50% increase in retinal intercellular adhesion molecule (ICAM)-1 expression and leukocyte accumulation, respectively, demonstrated a 2-fold increase in the levels of BM collagen IV and LOX, key determinants of capillary BM stiffness. Using atomic force microscopy, we confirmed that HG significantly enhances LOX-dependent subendothelial matrix stiffness in vitro, which correlated with an ∼2.5-fold increase in endothelial ICAM-1 expression, a 4-fold greater monocyte-EC adhesion, and an ∼2-fold alteration in endothelial NO (decrease) and NF-κB activation (increase). Inhibition of LOX-dependent subendothelial matrix stiffening alone suppressed HG-induced retinal EC activation. Finally, using synthetic matrices of tunable stiffness, we demonstrated that subendothelial matrix stiffening is necessary and sufficient to promote EC activation. These findings implicate BM stiffening as a critical determinant of HG-induced retinal EC activation and provide a rationale for examining BM stiffness and underlying mechanotransduction pathways as therapeutic targets for diabetic retinopathy.

Diabetic Macular Edema: Pathophysiology and Novel Therapeutic Targets.

Diabetic macular edema (DME) is the major cause of vision loss in diabetic persons. Alteration of the blood-retinal barrier is the hallmark of this disease, characterized by pericyte loss and endothelial cell-cell junction breakdown. Recent animal and clinical studies strongly indicate that DME is an inflammatory disease. Multiple cytokines and chemokines are involved in the pathogenesis of DME, with multiple cellular involvement affecting the neurovascular unit. With the introduction of anti-vascular endothelial growth factor (VEGF) agents, the treatment of DME has been revolutionized, and the indication for laser therapy has been limited. However, the response to anti-VEGF drugs in DME is not as robust as in proliferative diabetic retinopathy, and many patients with DME do not show complete resolution of fluid despite multiple intravitreal injections. Potential novel therapies targeting molecules other than VEGF and using new drug-delivery systems currently are being developed and evaluated in clinical trials.

Nuclear factor of activated T cells is activated in the endothelium of retinal microvessels in diabetic mice.

The pathogenesis of diabetic retinopathy (DR) remains unclear but hyperglycemia is an established risk factor. Endothelial dysfunction and changes in Ca2+ signaling have been shown to precede the onset of DR. We recently demonstrated that high extracellular glucose activates the Ca(2+)/calcineurin-dependent transcription factor NFAT in cerebral arteries and aorta, promoting the expression of inflammatory markers. Here we show, using confocal immunofluorescence, that NFAT is expressed in the endothelium of retinal microvessels and is readily activated by high glucose. This was inhibited by the NFAT blocker A-285222 as well as by the ectonucleotidase apyrase, suggesting a mechanism involving the release of extracellular nucleotides. Acute hyperglycemia induced by an IP-GTT (intraperitoneal glucose tolerance test) resulted in increased NFATc3 nuclear accumulation and NFAT-dependent transcriptional activity in retinal vessels of NFAT-luciferase reporter mice. In both Akita (Ins2(+/-) ) and streptozotocin- (STZ-) induced diabetic mice, NFAT transcriptional activity was elevated in retinal vessels. In vivo inhibition of NFAT with A-285222 decreased the expression of OPN and ICAM-1 mRNA in retinal vessels, prevented a diabetes driven downregulation of anti-inflammatory IL-10 in retina, and abrogated the increased vascular permeability observed in diabetic mice. Results identify NFAT signaling as a putative target for treatment of microvascular complications in diabetes.

Elevated cytokines, thrombin and PAI-1 in severe HCPS patients due to Sin Nombre virus.

Sin Nombre Hantavirus (SNV, Bunyaviridae Hantavirus) is a Category A pathogen that causes Hantavirus Cardiopulmonary Syndrome (HCPS) with case fatality ratios generally ranging from 30% to 50%. HCPS is characterized by vascular leakage due to dysregulation of the endothelial barrier function. The loss of vascular integrity results in non-cardiogenic pulmonary edema, shock, multi-organ failure and death. Using Electric Cell-substrate Impedance Sensing (ECIS) measurements, we found that plasma samples drawn from University of New Mexico Hospital patients with serologically-confirmed HCPS, induce loss of cell-cell adhesion in confluent epithelial and endothelial cell monolayers grown in ECIS cultureware. We show that the loss of cell-cell adhesion is sensitive to both thrombin and plasmin inhibitors in mild cases, and to thrombin only inhibition in severe cases, suggesting an increasing prothrombotic state with disease severity. A proteomic profile (2D gel electrophoresis and mass spectrometry) of HCPS plasma samples in our cohort revealed robust antifibrinolytic activity among terminal case patients. The prothrombotic activity is highlighted by acute ≥30 to >100 fold increases in active plasminogen activator inhibitor (PAI-1) which, preceded death of the subjects within 48 h. Taken together, this suggests that PAI-1 might be a response to the severe pathology as it is expected to reduce plasmin activity and possibly thrombin activity in the terminal patients.

Chemokine mediated monocyte trafficking into the retina: role of inflammation in alteration of the blood-retinal barrier in diabetic retinopathy.

Inflammation in the diabetic retina is mediated by leukocyte adhesion to the retinal vasculature and alteration of the blood-retinal barrier (BRB). We investigated the role of chemokines in the alteration of the BRB in diabetes. Animals were made diabetic by streptozotocin injection and analyzed for gene expression and monocyte/macrophage infiltration. The expression of CCL2 (chemokine ligand 2) was significantly up-regulated in the retinas of rats with 4 and 8 weeks of diabetes and also in human retinal endothelial cells treated with high glucose and glucose flux. Additionally, diabetes or intraocular injection of recombinant CCL2 resulted in increased expression of the macrophage marker, F4/80. Cell culture impedance sensing studies showed that purified CCL2 was unable to alter the integrity of the human retinal endothelial cell barrier, whereas monocyte conditioned medium resulted in significant reduction in cell resistance, suggesting the relevance of CCL2 in early immune cell recruitment for subsequent barrier alterations. Further, using Cx3cr1-GFP mice, we found that intraocular injection of CCL2 increased retinal GFP+ monocyte/macrophage infiltration. When these mice were made diabetic, increased infiltration of monocytes/macrophages was also present in retinal tissues. Diabetes and CCL2 injection also induced activation of retinal microglia in these animals. Quantification by flow cytometry demonstrated a two-fold increase of CX3CR1+/CD11b+ (monocyte/macrophage and microglia) cells in retinas of wildtype diabetic animals in comparison to control non-diabetic ones. Using CCL2 knockout (Ccl2-/-) mice, we show a significant reduction in retinal vascular leakage and monocyte infiltration following induction of diabetes indicating the importance of this chemokine in alteration of the BRB. Thus, CCL2 may be an important therapeutic target for the treatment of diabetic macular edema.

The role of monocyte subsets in myocutaneous revascularization.

BACKGROUND: The controlled recruitment of monocytes from the circulation to the site of injury and their differentiation into tissue macrophages are critical events in the reconstitution of tissue integrity. Subsets of monocytes/macrophages have been implicated in the pathogenesis of atherosclerosis and tumor vascularity; however, the significance of monocyte heterogeneity in physiologic neovascularization is just emerging. MATERIALS AND METHODS: A cranial-based, peninsular-shaped myocutaneous flap was surgically created on the dorsum of wild-type mice (C57BL6) and populations of mice with genetic deletion of subset-specific chemokine ligand-receptor axes important in monocyte trafficking and function (CCL2(-/-) and CX3CR1(-/-)) (n=36 total; 12 mice per group, nine with flap and three unoperated controls). Planimetric analysis of digital photographic images was utilized to determine flap surface viability in wild-type and knockout mice. Real-time myocutaneous flap perfusion and functional revascularization was determined by laser speckle contrast imaging. Image analysis of CD-31 immunostained sections confirmed flap microvascular density and anatomy. Macrophage quantification and localization in flap tissues was determined by F4/80 gene and protein expression. Quantitative reverse transcription-polymerase chain reaction was performed on nonoperative back skin and postoperative flap tissue specimens to determine local gene expression. RESULTS: Myocutaneous flaps created on wild type and CX3CR1(-/-) mice were engrafted to the recipient site, resulting in viability. In contrast, distal full thickness cutaneous necrosis and resultant flap dehiscence was evident by d 10 in CCL2(-/-) mice. Over 10 d, laser speckle contrast imaging documented immediate graded flap ischemia in all three groups of mice, functional flap revascularization in wild type and CX3CR1(-/-) mice, and lack of distal flap reperfusion in CCL2(-/-) mice. Immunostaining of serial histologic specimens confirmed marked increases in microvascular density and number of macrophages in wild type mice, intermediate increases in CX3CR1(-/-) mice, and no significant change in vessel count or macrophage quantity in CCL2(-/-) mice over the study interval. Finally, quantitative reverse transcriptase polymerase chain reaction demonstrated that the loss of function of chemokine ligand and receptor genes influenced the transcription of local genes involved in monocyte chemotaxis and wound angiogenesis. CONCLUSIONS: In a graded-ischemia wound healing model, monocyte recruitment was severely impaired in CCL2(-/-) mice, resulting in failure of flap revascularization and concomitant cutaneous necrosis. Analysis of CX3CR1-deficient mice revealed adequate monocyte recruitment and revascularization for flap survival; however, the myeloid cell response and magnitude of neovascularization were dampened compared with wild type mice.

Diabetic retinopathy and inflammation: novel therapeutic targets.

Most anti-vascular endothelial growth factor (VEGF) therapies in diabetic macular edema are not as robust as in proliferative diabetic retinopathy. Although the VEGF appears to be a good target in diabetic macular edema, the anti-VEGF therapies appear to be of transient benefit as the edema recurs within a few weeks, and repeated injections are necessary. There is new evidence that indicates 'retinal inflammation' as an important player in the pathogenesis of diabetic retinopathy. There are common sets of inflammatory cytokines that are upregulated in both the serum and vitreous and aqueous samples, in subjects with diabetic retinopathy, and these cytokines can have multiple interactions to impact the pathogenesis of the disease. The key inflammatory events involved in the blood retinal barrier (BRB) alteration appear to be: (1) Increased expression of endothelial adhesion molecules such as ICAM1, VCAM1, PECAM-1, and P-selectin, (2) adhesion of leukocytes to the endothelium, (3) release of inflammatory chemokines, cytokines, and vascular permeability factors, (4) alteration of adherens and tight junctional proteins between the endothelial cells, and (5) infiltration of leukocytes into the neuro-retina, resulting in the alteration of the blood retinal barrier (diapedesis). VEGF inhibition itself may not achieve neutralization of other inflammatory molecules involved in the inflammatory cascade of the breakdown of the BRB. It is possible that the novel selective inhibitors of the inflammatory cascade (like angiopoietin-2, TNFα, and chemokines) may be useful therapeutic agents in the treatment of diabetic macular edema (DME), either alone or in combination with the anti-VEGF drugs.

Pattern recognition receptor gene expression in ischemia-induced flap revascularization.

BACKGROUND: The innate immune system is the major contributor to acute inflammation induced by microbial infection or tissue damage. Germline-encoded pattern recognition receptors (PRRs) are responsible for sensing the presence of micro-organisms and endogenous molecules released from damaged cells. We performed microarray analyses on ischemic wound tissue to investigate the temporal relationship between PRR gene expression, wound perfusion, and flap revascularization. METHODS: A cranial-based, peninsular-shaped myocutaneous flap was surgically created on the dorsum of C57BL6 mice (n = 25 total; n = 20 with flap). Laser speckle contrast imaging was utilized to study the pattern of flap ischemia and return of functional revascularization. Flap microvascular density was determined by image analysis of CD-31-immunostained sections. Total RNA was isolated from homogenized flap tissue and was converted to cDNA (RT), which was hybridized to a microarray of pathway-focused genes. Microarray results were validated with quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR). RESULTS: Laser speckle contrast imaging predicted the spatial and temporal pattern of ischemia and functional revascularization. Histologic analysis demonstrated early leukocyte infiltration and later engraftment, resulting in flap revascularization by new blood vessel growth from the recipient bed and dilatation of preexisting proximal flap vasculature. qRT-PCR demonstrated significant early gene expression of select PRRs, cytokines, chemokines, and growth factors, peaking by 48 hours, and returning toward baseline but remaining elevated at 10 days. CONCLUSION: Surgical and ischemic tissue injury resulted in the early gene expression of select PRRs, which may bind with endogenous molecules released from ischemic or necrotic cells, leading to transcription of genes involved in wound inflammation and angiogenesis.

Pericyte-derived sphingosine 1-phosphate induces the expression of adhesion proteins and modulates the retinal endothelial cell barrier.

OBJECTIVE: The mechanisms that regulate the physical interaction of pericytes and endothelial cells and the effects of these interactions on interendothelial cell junctions are not well understood. We determined the extent to which vascular pericytes could regulate pericyte-endothelial adhesion and the consequences that this disruption might have on the function of the endothelial barrier. METHODS AND RESULTS: Human retinal microvascular endothelial cells were cocultured with pericytes, and the effect on the monolayer resistance of endothelial cells and expression of the cell junction molecules N-cadherin and VE-cadherin were measured. The molecules responsible for the effect of pericytes or pericyte-conditioned media on the endothelial resistance and cell junction molecules were further analyzed. Our results indicate that pericytes increase the barrier properties of endothelial cell monolayers. This barrier function is maintained through the secretion of pericyte-derived sphingosine 1-phosphate. Sphingosine 1-phosphate aids in maintenance of microvascular stability by upregulating the expression of N-cadherin and VE-cadherin, and downregulating the expression of angiopoietin 2. CONCLUSIONS: Under normal circumstances, the retinal vascular pericytes maintain pericyte-endothelial contacts and vascular barrier function through the secretion of sphingosine 1-phosphate. Alteration of pericyte-derived sphingosine 1-phosphate production may be an important mechanism in the development of diseases characterized by vascular dysfunction and increased permeability.