[Effect on the pain and tear film stability in patients after pterygium excision treated with intradermal needling].

OBJECTIVE: To analyze the effects of intradermal needling for pain and tear film stability in patients after pterygium excision. METHODS: A total of 76 patients (98 affected eyes) with primary pterygium were randomly divided into an observation group (38 cases, 53 affected eyes) and a control group (38 cases, 45 affected eyes).In the control group, only pterygium resection was performed, in the observation group, intradermal needling after pterygium resection was applied at Cuanzhu (BL 2), Yuyao (EX-HN 4), Taiyang (EX-HN 5), Sibai (ST 2), Hegu (LI 4), removed after 24 h and changed three times a week. The pain level of 3 days after surgery, dry eye symptoms, the basic tear secretion test (Schirmer-Ⅰ), and the tear-break time (BUT) changes before surgery, 2 weeks after surgery and 4 weeks after surgery were compared between the two groups, and the clinical efficacy was evaluated. RESULTS: The pain level of 3 days after surgery in the observation group was significantly lower than that in the control group (P<0.05). The dry eye symptom scores at 2 weeks and 4 weeks after surgery in the two groups were significantly lower than those before surgery (all P<0.05), and the dry eye symptom scores in the observation group were significantly lower than those in the control group (both P<0.05). The Schirmer-Ⅰ test at 2 weeks and 4 weeks after surgery was significantly prolonged than that before surgery(all P<0.05), and the Schirmer-Ⅰ test in the observation group was significantly longer than that in the control group (both P<0.05). The BUT at 2 weeks and 4 weeks after surgery in the two groups was significantly longer than that before surgery (all P<0.05), and the BUT in the observation group was significantly longer than that in the control group (both P<0.05). The total effective rate in the observation group was 89.5% (34/38), which was higher than 71.1% (27/38) in the control group (P<0.05). CONCLUSION: Intradermal needling can effectively reduce the pain level of patients after pterygium resection, improve dry eye symptoms, promote the secretion of tears and improve the tear film stability.

CCL19-CCR7-dependent reverse transendothelial migration of myeloid cells clears Chlamydia muridarum from the arterial intima.

Regions of the normal arterial intima predisposed to atherosclerosis are sites of ongoing monocyte trafficking and also contain resident myeloid cells with features of dendritic cells. However, the pathophysiological roles of these cells are poorly understood. Here we found that intimal myeloid cells underwent reverse transendothelial migration (RTM) into the arterial circulation after systemic stimulation of pattern-recognition receptors (PRRs). This process was dependent on expression of the chemokine receptor CCR7 and its ligand CCL19 by intimal myeloid cells. In mice infected with the intracellular pathogen Chlamydia muridarum, blood monocytes disseminated infection to the intima. Subsequent CCL19-CCR7-dependent RTM was critical for the clearance of intimal C. muridarum. This process was inhibited by hypercholesterolemia. Thus, RTM protects the normal arterial intima, and compromised RTM during atherogenesis might contribute to the intracellular retention of pathogens in atherosclerotic lesions.

A novel assay uncovers an unexpected role for SR-BI in LDL transcytosis.

AIMS: Retention of low-density lipoprotein (LDL) cholesterol beneath the arterial endothelium initiates an inflammatory response culminating in atherosclerosis. Since the overlying endothelium is healthy and intact early on, it is likely that LDL passes through endothelial cells by transcytosis. However, technical challenges have made confirming this notion and elucidating the mechanisms of transcytosis difficult. We developed a novel assay for measuring LDL transcytosis in real time across coronary endothelial cell monolayers; we used this approach to identify the receptor involved. METHODS AND RESULTS: Murine aortas were perfused ex vivo with LDL and dextran of a smaller molecular radius. LDL (but not dextran) accumulated under the endothelium, indicating that LDL transcytosis occurs in intact vessels. We then confirmed that LDL transcytosis occurs in vitro using human coronary artery endothelial cells. An assay was developed to quantify transcytosis of DiI-LDL in real time using total internal reflection fluorescence microscopy. DiI-LDL transcytosis was inhibited by excess unlabelled LDL, while degradation of the LDL receptor by PCSK9 had no effect. Instead, LDL colocalized partially with the scavenger receptor SR-BI and overexpression of SR-BI increased LDL transcytosis; knockdown by siRNA significantly reduced it. Excess HDL, the canonical SR-BI ligand, significantly decreased LDL transcytosis. Aortas from SR-BI-deficient mice were perfused ex vivo with LDL and accumulated significantly less sub-endothelial LDL compared with wild-type littermates. CONCLUSION: We developed an assay to quantify LDL transcytosis across endothelial cells and discovered an unexpected role for SR-BI. Elucidating the mechanisms of LDL transcytosis may identify novel targets for the prevention or therapy of atherosclerosis.

Differential role of an NF-κB transcriptional response element in endothelial versus intimal cell VCAM-1 expression.

RATIONALE: Human and murine Vcam1 promoters contain 2 adjacent nuclear factor-κB (NF-κB)-binding elements. Both are essential for cytokine-induced transcription of transiently transfected promoter-reporter constructs. However, the relevance of these insights to regulation of the endogenous Vcam1 gene and to pathophysiological processes in vivo remained unknown. OBJECTIVE: Determine the role of the 5' NF-κB-binding element in expression of the endogenous Vcam1 gene. METHODS AND RESULTS: Homologous recombination in embryonic stem cells was used to inactivate the 5' NF-κB element in the Vcam1 promoter and alter 3 nucleotides in the 5' untranslated region to allow direct comparison of wild-type versus mutant allele RNA expression and chromatin configuration in heterozygous mice. Systemic treatment with inflammatory cytokines or endotoxin (lipopolysaccharide) induced lower expression of the mutant allele relative to wild-type by endothelial cells in the aorta, heart, and lungs. The mutant allele also showed lower endothelial expression in 2-week atherosclerotic lesions in Vcam1 heterozygous/low-density lipoprotein receptor-deficient mice fed a cholesterol-rich diet. In vivo chromatin immunoprecipitation assays of heart showed diminished lipopolysaccharide-induced association of RNA polymerase 2 and NF-κB p65 with the mutant promoter. In contrast, expression of mutant and wild-type alleles was comparable in intimal cells of wire-injured carotid artery and 4- to 12-week atherosclerotic lesions. CONCLUSIONS: This study highlights differences between in vivo and in vitro promoter analyses, and reveals a differential role for a NF-κB transcriptional response element in endothelial vascular cell adhesion molecule-1 expression induced by inflammatory cytokines or a cholesterol-rich diet versus intimal cell expression in atherosclerotic lesions and injured arteries.

Aortic regurgitation dramatically alters the distribution of atherosclerotic lesions and enhances atherogenesis in mice.

OBJECTIVE: Hemodynamics plays a critical role in atherogenesis, but the association between flow pattern and preferential localization of lesion is not fully understood. We developed a mouse model of aortic valve regurgitation (AR) to change the aortic flow pattern and observed the effects on plaque formation. METHODS AND RESULTS: High-frequency Doppler ultrasound imaging of 10 untreated C57BL/6J mice and 6 sham-treated low-density lipoprotein receptor-deficient (Ldlr(-/-)) mice revealed consistent antegrade blood flow throughout the aorta and oscillatory flow only along the lesser curvature of the aortic arch. Catheter-induced AR in 7 Ldlr(-/-) mice produced various degrees of diastolic retrograde flow throughout the aorta. After the mice were fed a cholesterol-enriched diet for 6 weeks, the burden of atherosclerotic lesions was increased 6-fold, with the naturally plaque-resistant descending aorta becoming susceptible. The AR severity correlated positively with the lesion burden in the descending thoracic and abdominal aorta but negatively with the lesions in the ascending aorta and aortic arch. CONCLUSIONS: This AR model is valuable for elucidating the relationship between hemodynamics and predisposition of the artery wall to atherosclerosis, because of the significant alterations in local flow patterns and the conversion of large regions in the descending aorta from lesion resistant to lesion prone.

Resident intimal dendritic cells accumulate lipid and contribute to the initiation of atherosclerosis.

RATIONALE: Atherosclerosis is an inflammatory disease in which leukocytes and oxidatively modified lipids accumulate in the arterial intima. Previously, we showed that dendritic cells (DCs) accumulate preferentially in regions predisposed to atherosclerosis in the normal murine aortic intima. The function of these cells in atherogenesis is unknown. OBJECTIVE: Our goal was to determine the role of resident intimal DCs in the initiation of atherosclerosis. METHODS AND RESULTS: En face immunostaining of nascent atherosclerotic lesions in low-density lipoprotein receptor-deficient (Ldlr(-/-)) mice fed a cholesterol-rich diet for 5 or 10 days demonstrated that foam cells expressed DC markers CD11c, 33D1, and major histocompatibility complex class II. Transmission electron microscopy revealed that the majority of intimal lipid was intracellular. The role of resident intimal DCs in lesion formation was verified by their conditional depletion using transgenic mice expressing the simian diphtheria toxin receptor in CD11c(+) cells. A single injection of diphtheria toxin depleted intimal CD11c(+) DCs by >98% within 24 hours, with 25% and 75% recovery at 1 and 3 weeks, respectively. When bred onto the Ldlr(-/-) background, intimal DC depletion with diphtheria toxin during 5 days of lesion formation reduced the intimal lipid area by 55% relative to undepleted controls. Transmission electron microscopy revealed few foam cells in DC-depleted mice and abundant accumulation of subendothelial extracellular lipid. CONCLUSIONS: Induction of hypercholesterolemia in mice triggers rapid ingestion of lipid by resident intimal DCs, which initiate nascent foam cell lesion formation.

Discoidin domain receptor 1 on bone marrow-derived cells promotes macrophage accumulation during atherogenesis.

RATIONALE: We described a critical role for the discoidin domain receptor (DDR)1 collagen receptor tyrosine kinase during atherosclerotic plaque development. Systemic deletion of Ddr1 in Ldlr(-/-) mice accelerated matrix accumulation and reduced plaque size and macrophage content. However, whether these effects reflected an independent role for macrophage DDR1 during atherogenesis remained unresolved. METHODS: In the present study, we performed sex-mismatched bone marrow transplantation using Ddr1(+/+);Ldlr(-/-) and Ddr1(-/-);Ldlr(-/-) mice to investigate the role of macrophage DDR1 during atherogenesis. Chimeric mice with deficiency of DDR1 in bone marrow-derived cells (Ddr1(-/--->+/+)) or control chimeric mice that received Ddr1(+/+);Ldlr(-/-) marrow (Ddr1(+/+-->+/+)) were fed an atherogenic diet for 12 weeks. RESULTS: We observed a 66% reduction in atherosclerosis in the descending aorta and a 44% reduction in plaque area in the aortic sinus in Ddr1(-/--->+/+) mice compared to Ddr1(+/+-->+/+) mice. Furthermore, we observed a specific reduction in the number of donor-derived macrophages in Ddr1(-/--->+/+) plaques, suggesting that bone marrow deficiency of DDR1 attenuated atherogenesis by limiting macrophage accumulation in the plaque. We have also demonstrated that the effects of DDR1 on macrophage infiltration and accumulation can occur at the earliest stage of atherogenesis, the formation of the fatty streak. Deficiency of DDR1 limited the appearance of 5-bromodeoxyuridine-labeled monocytes/macrophages in the fatty streak and resulted in reduced lesion size in Ldlr(-/-) mice fed a high fat diet for 2 weeks. In vitro studies to investigate the mechanisms involved revealed that macrophages from Ddr1(-/-) mice had decreased adhesion to type IV collagen and decreased chemotactic invasion of type IV collagen in response to monocyte chemoattractant protein-1. CONCLUSIONS: Taken together, our data support an independent and critical role for DDR1 in macrophage accumulation at early and late stages of atherogenesis.

GM-CSF regulates intimal cell proliferation in nascent atherosclerotic lesions.

The contribution of intimal cell proliferation to the formation of early atherosclerotic lesions is poorly understood. We combined 5-bromo-2'-deoxyuridine pulse labeling with sensitive en face immunoconfocal microscopy analysis, and quantified intimal cell proliferation and Ly-6C(high) monocyte recruitment in low density lipoprotein receptor-null mice. Cell proliferation begins in nascent lesions preferentially at their periphery, and proliferating cells accumulate in lesions over time. Although intimal cell proliferation increases in parallel to monocyte recruitment as lesions grow, proliferation continues when monocyte recruitment is inhibited. The majority of proliferating intimal cells are dendritic cells expressing CD11c and major histocompatibility complex class II and 33D1, but not CD11b. Systemic injection of granulocyte/macrophage colony-stimulating factor (GM-CSF) markedly increased cell proliferation in early lesions, whereas function-blocking anti-GM-CSF antibody inhibited proliferation. These findings establish GM-CSF as a key regulator of intimal cell proliferation in lesions, and demonstrate that both proliferation and monocyte recruitment contribute to the inception of atherosclerosis.

Endothelial nitric oxide synthase gene expression during murine embryogenesis: commencement of expression in the embryo occurs with the establishment of a unidirectional circulatory system.

To elucidate the role of endothelial NO synthase (eNOS)-derived NO during mammalian embryogenesis, we assessed the expression of the eNOS gene during development. Using transgenic eNOS promoter/reporter mice (with beta-galactosidase and green fluorescent protein reporters), in situ cRNA hybridization, and immunohistochemistry to assess transcription, steady-state mRNA levels, and protein expression, respectively, we noted that eNOS expression in the developing cardiovascular system was highly restricted to endothelial cells of medium- and large-sized arteries and the endocardium. The onset of transcription of the native eNOS gene and reporters coincided with the establishment of robust, unidirectional blood flow at embryonic day 9.5, as assessed by Doppler ultrasound biomicroscopy. Interestingly, reporter transgene expression and native eNOS mRNA were also observed in discrete regions of the developing skeletal musculature and the apical ectodermal ridge of developing limbs, suggesting a role for eNOS-derived NO in limb development. In vitro studies of promoter/reporter constructs indicated that similar eNOS promoter regions operate in both embryonic skeletal muscle and vascular endothelial cells. In summary, transcriptional activity of the eNOS gene in the murine circulatory system occurred following the establishment of embryonic blood flow. Thus, the eNOS gene is a late-onset gene in endothelial ontogeny.

Relative reduction of endothelial nitric-oxide synthase expression and transcription in atherosclerosis-prone regions of the mouse aorta and in an in vitro model of disturbed flow.

Atherosclerosis develops in distinct regions of the arterial tree. Defining patterns and mechanisms of endothelial cell gene expression in different regions of normal arteries is key to understanding the initial molecular events in atherogenesis. In this study, we demonstrated that the expression of endothelial nitric-oxide synthase (eNOS), an atheroprotective gene, and its phosphorylation on Ser(1177), a marker of activity, were lower in regions of the normal mouse aorta that are predisposed to atherosclerosis. The same expression pattern was observed in mouse strains that are both susceptible and resistant to atherosclerosis, and the topography of eNOS expression was inverse to p65, the main nuclear factor-kappaB subunit. Modeling of disturbed and uniform laminar flow in vitro reproduced the expression patterns of eNOS and p65 that were found in vivo. Heterogeneous nuclear RNA expression and RNA polymerase II chromosome immunoprecipitation studies demonstrated that regulation of transcription contributed to increased eNOS expression in response to shear stress. In vivo, the transcription of eNOS was reduced in regions of the mouse aorta predisposed to atherosclerosis, as defined by reporter gene expression in eNOS promoter-beta-galactosidase reporter transgenic mice. These data suggest that disturbed hemodynamic patterns found at arterial branches and curvatures uniquely modulate endothelial cell gene expression by regulating transcription, potentially explaining why these regions preferentially develop atherosclerosis when risk factors such as hypercholesterolemia are introduced.

Low-grade chronic inflammation in regions of the normal mouse arterial intima predisposed to atherosclerosis.

Atherosclerotic lesions develop in regions of arterial curvature and branch points, which are exposed to disturbed blood flow and have unique gene expression patterns. The cellular and molecular basis for atherosclerosis susceptibility in these regions is not completely understood. In the intima of atherosclerosis-predisposed regions of the wild-type C57BL/6 mouse aorta, we quantified increased expression of several proinflammatory genes that have been implicated in atherogenesis, including vascular cell adhesion molecule-1 (VCAM-1) and a relative abundance of dendritic cells, but only occasional T cells. In contrast, very few intimal leukocytes were detected in regions resistant to atherosclerosis; however, abundant macrophages, including T cells, were found throughout the adventitia (Adv). Considerably lower numbers of intimal CD68+ leukocytes were found in inbred atherosclerosis-resistant C3H and BALB/c mouse strains relative to C57BL/6 and 129; however, leukocyte distribution throughout the Adv of all strains was similar. The predominant mechanism for the accumulation of intimal CD68+ cells was continued recruitment of bone marrow-derived blood monocytes, suggestive of low-grade chronic inflammation. Local proliferation of intimal leukocytes was low. Intimal CD68+ leukocytes were reduced in VCAM-1-deficient mice, suggesting that mechanisms of leukocyte accumulation in the intima of normal aorta are analogous to those in atherosclerosis.