Heparin-binding EGF-like growth factor (HB-EGF) antisense oligonucleotide protected against hyperlipidemia-associated atherosclerosis.

BACKGROUND AND AIMS: Heparin-binding EGF-like growth factor (HB-EGF) is a representative EGF family member that interacts with EGFR under diverse stress environment. Previously, we reported that the HB-EGF-targeting using antisense oligonucleotide (ASO) effectively suppressed an aortic aneurysm in the vessel wall and circulatory lipid levels. In this study, we further examined the effects of the HB-EGF ASO administration on the development of hyperlipidemia-associated atherosclerosis using an atherogenic mouse model. METHODS AND RESULTS: The male and female LDLR deficient mice under Western diet containing 21% fat and 0.2% cholesterol content were cotreated with control and HB-EGF ASOs for 12 weeks. We observed that the HB-EGF ASO administration effectively downregulated circulatory VLDL- and LDL-associated lipid levels in circulation; concordantly, the HB-EGF targeting effectively suppressed the development of atherosclerosis in the aorta. An EGFR blocker BIBX1382 administration suppressed the hepatic TG secretion rate, suggesting a positive role of the HB-EGF signaling for the hepatic VLDL production. We newly observed that there was a significant improvement of the insulin sensitivity by the HB-EGF ASO administration in a mouse model under the Western diet as demonstrated by the improvement of the glucose and insulin tolerances. CONCLUSION: The HB-EGF ASO administration effectively downregulated circulatory lipid levels by suppressing hepatic VLDL production rate, which leads to effective protection against atherosclerosis in the vascular wall.

Non-target effect of continuous application of chlorpyrifos on soil microbes, nematodes and its persistence under sub-humid tropical rice-rice cropping system.

Application of pesticide in agricultural fields is "unnecessary evil" for non-target microflora and fauna. Hence, to identify the safer pesticide molecules against non-target microbes, a long-term pesticide experiment was initiated at National Rice Research Institute, Cuttack, India. In the present study, the effect of continuous application of chlorpyrifos (0.5kgha-1) in rice fields on non-target groups of soil microbes and nematodes was studied for seven seasons (four wet and three dry seasons) during 2009-2013. Treatments were arranged in a randomized complete block design with four replications of chlorpyrifos-treated (0.5kg a.i. ha-1) (CT) and untreated control (UT) plots. During seven seasons of experimentation, regular application of chlorpyrifos had no significant effect on population of heterotrophic aerobic, anaerobic, oligotrophic and copiotrophic bacteria in CT compared to UT, whereas, population of asymbiotic aerobic nitrogen fixer, nitrifiers, denitrifiers, gram positive and spore-forming bacteria were significantly reduced by nearly 0.25-2 fold in CT than UT. However, comparatively less deviation in population of actinomycetes, fungi, phosphate solubilizing and sulfur oxidizing bacteria were observed in CT than UT. Significant interactions were found between effects of chlorpyrifos with time in population dynamics of microbes. In plant parasitic nematode species, Meloidogyne graminicola (RRKN) and Hirschmanniella spp. (RRN), were significantly lower (p<0.01) in CT compared to UT after first year onwards. The overall observation of five years data indicated that the RRKN population showed a decreasing trend (R2=0.644) whereas RRN showed increasing trend (R2=0.932) in CT. The drastic chlorpyrifos dissipation was noticed after 15 days of application from the initial residue of 0.25mgkg-1 soil, which indicated that chlorpyrifos residue in rice field soil was not persistent and its half-life was found to be 4.02 days. Overall, the present findings revealed that non-target effect of repetitive application of chloropyrifos (0.5kgha-1) on soil microbes and nematodes was found less under rice-rice cropping system.

Effect of Pretilachlor on Soil Enzyme Activities in Tropical Rice Soil.

Pretilachlor treatments, namely, recommended dose at 600 g a.i. ha-1 (RD), double the recommended dose at 1200 g a.i. ha-1 (2RD), ten times of the recommended dose at 6000 g a.i. ha-1 (10RD) along with control, were used to study the effects of pretilachlor on soil enzymes in tropical rice soil. Pretilachlor, at recommended dose completely dissipated 30 days after herbicide application. Twenty days after herbicide application, the dehydrogenase activity was inhibited up to 27 %, 28 % and 40 % of initial values of RD, 2RD and 10RD treatments, respectively. Increase in fluorescein diacetate hydrolase activity was observed during the first 25 days post herbicide application up to 29 %, 36 % and 10 % of initial values of RD, 2RD and 10RD treatments, respectively. ß-Glucosidase activity in the experiment did not provide a specific trend. In general, urease and acid phosphatase activities were not influenced by pretilachlor application. There were significant differences in alkaline phosphatase activities among the treatments until 25 days after herbicide application. Hence, pretilachlor may cause short term transitory changes in soil enzyme parameters. However, it has negative impact on soil enzymes at very high dose.

Effect of elevated CO2 on chlorpyriphos degradation and soil microbial activities in tropical rice soil.

Impact of elevated CO2 on chlorpyriphos degradation, microbial biomass carbon, and enzymatic activities in rice soil was investigated. Rice (variety Naveen, Indica type) was grown under four conditions, namely, chambered control, elevated CO2 (550 ppm), elevated CO2 (700 ppm) in open-top chambers and open field. Chlorpyriphos was sprayed at 500 g a.i. ha(-1) at maximum tillering stage. Chlorpyriphos degraded rapidly from rice soils, and 88.4% of initially applied chlorpyriphos was lost from the rice soil maintained under elevated CO2 (700 ppm) by day 5 of spray, whereas the loss was 80.7% from open field rice soil. Half-life values of chlorpyriphos under different conditions ranged from 2.4 to 1.7 days with minimum half-life recorded with two elevated CO2 treatments. Increased CO2 concentration led to increase in temperature (1.2 to 1.8 °C) that played a critical role in chlorpyriphos persistence. Microbial biomass carbon and soil enzymatic activities specifically, dehydrogenase, fluorescien diacetate hydrolase, urease, acid phosphatase, and alkaline phosphatase responded positively to elevated CO2 concentrations. Generally, the enzyme activities were highly correlated with each other. Irrespective of the level of CO2, short-term negative influence of chlorpyriphos was observed on soil enzymes till day 7 of spray. Knowledge obtained from this study highlights that the elevated CO2 may negatively influence persistence of pesticide but will have positive effects on soil enzyme activities.

Needs of an uninsured equity-deserving minority patient cohort with physical disabilities during the first wave of the COVID-19 pandemic.

Background: Patients with disabilities and those from diverse equity-deserving backgrounds have been disproportionately affected by the SARS COV-2 ("COVID-19") pandemic. Objective: To describe the significant needs and social determinants of health that affected a group of uninsured patients (from equity-deserving groups) with rehabilitation diagnoses during the early months of the COVID-19 pandemic. Design: Retrospective cohort study utilizing a telephone-based needs assessment from April to October, 2020. Setting: Free interdisciplinary rehabilitation clinic serving patients with physical disabilities from equity-deserving minority backgrounds. Participants: 51 uninsured, diverse patients with spinal cord injuries, brain injuries, amputations, strokes, and other diagnoses requiring interdisciplinary rehabilitation care. Methods: Using a non-structured approach, telephone-based needs assessments were collected monthly. Reported needs were summarized into themes and the frequencies of each theme were recorded. Results: From the total number of concerns, medical issues were reported with the highest frequency (46%), followed by equipment needs (30%) and mental health concerns (30%). Other frequently mentioned needs centered around themes of rent, employment, and supplies. Rent and employment were more frequently cited in earlier months, and equipment problems were more frequently cited in later months. A minority of patients reported they had no needs, some of whom had acquired insurance. Conclusions: Our objective was to describe the needs of a racially and ethnically diverse set of uninsured individuals with physical disabilities seen at a specialized interdisciplinary rehabilitation pro bono clinic during the early months of COVID-19. Medical issues, equipment needs, and mental health concerns were the top three needs. To optimally serve them, care providers must be aware of current and future needs for their underserved patients, especially if future lockdowns occur.

Novel vascular molecule involved in monocyte adhesion to aortic endothelium in models of atherogenesis.

Adhesion of monocytes to the endothelium in lesion-prone areas is one of the earliest events in fatty streak formation leading to atherogenesis. The molecular basis of increased monocyte adhesion is not fully characterized. We have identified a novel vascular monocyte adhesion-associated protein, VMAP-1, that plays a role in adhesion of monocytes to activated endothelium. Originally selected for its ability to block binding of a mouse monocyte-like cell line (WEHI78/24) to cytokine- or LPS-stimulated cultured mouse endothelial cells in vitro, antiVMAP-1 mAb LM151 cross-reacts with rabbit endothelium and blocks binding of human monocytes to cultured rabbit aortic endothelial cells stimulated with minimally modified low density lipoprotein, thought to be a physiologically relevant atherogenic stimulus. Most importantly, LM151 prevents adhesion of normal monocytes and monocytoid cells to intact aortic endothelium from cholesterol-fed rabbits in an ex vivo assay. VMAP-1 is a 50-kD protein. Immunohistology of vessels reveals focal constitutive expression in aorta and other large vessels. VMAP-1 is thus a novel vascular adhesion-associated protein that appears to play a critical role in monocyte adhesion to aortic endothelial cells in atherogenesis in vivo.

Formation of enlarged mitochondria in a liver cell line in response to a synthetic glucocorticoid.

For a number of years it has been recognized that glucocorticoids cause alterations in liver cell morphology (6, 9). Several investigators have shown that in liver in vivo mitochondria can be enlarged to many times their normal volume by treatment with cortisone (13, 15). There is a concomitant decrease in mitochondrial number, and the results of Kimberg and Loeb suggest that this is due to mitochondrial fusion (7). However, the exact mechanism whereby mitochondrial volume is altered and whether in fact cortisone is the direct causal agent are not known due to the complexity of studying these questions in a whole animal system. We have found that dexamethasone sodium phosphate (dex), a synthetic glucocorticoid, causes the formation of enlarged mitochondria in a liver cell line RLC-GAI, which grows in defined medium. In this paper we present our observations on the amount of enlargement that occurs after 5 days of treatment. The formation of enlarged mitochondria is reversible upon removal of the hormone from the medium, and we have attempted to determine whether "mitochondrial" or "nonmitochondrial" inhibitors are more effective in blocking the return of mitochondria to their normal size when the hormone is removed.

Chlamydomonas flagella. I. Isolation and electrophoretic analysis of microtubules, matrix, membranes, and mastigonemes.

Methods were developed for the isolation of Chlamydomonas flagella and for their fractionation into membrane, mastigoneme, "matrix," and axoneme components. Each component was studied by electron microscopy and acrylamide gel electrophoresis. Purified membranes retained their tripartite ultrastructure and were shown to contain one high molecular weight protein band on electrophoresis in sodium dodecyl sulfate (SDS)-urea gels. Isolated mastigonemes (hairlike structures which extend laterally from the flagellar membrane in situ) were of uniform size and were constructed of ellipsoidal subunits joined end to end. Electrophoretic analysis of mastigonemes indicated that they contained a single glycoprotein of approximately 170,000 daltons The matrix fraction contained a number of proteins (particularly those of the amorphous material surrounding the microtubules), which became solubilized during membrane removal. Isolated axonemes retained the intact "9 + 2" microtubular structure and could be subfractionated by treatment with heat or detergent. Increasing concentrations of detergent solubilized axonemal microtubules in the following order: one of the two central tubules; the remaining central tubule and the outer wall of the B tubule; the remaining portions of the B tubule; the outer wall of the A tubule; the remainder of the A tubule with the exception of a ribbon of three protofilaments. These three protofilaments appeared to be the "partition" between the lumen of the A and B tubule. Electrophoretic analysis of isolated outer doublets of 9 + 2 flagella of wild-type cells and of "9 + 0" flagella of paralyzed mutants indicated that the outer doublets and central tubules were composed of two microtubule proteins (tubulins 1 and 2) Tubulins 1 and 2 were shown to have apparent molecular weights of 56,000 and 53,000 respectively

Magnetic resonance imaging of biological specimens by electron paramagnetic resonance of nitroxide spin labels.

Electron paramagnetic resonance imaging was demonstrated on two plant species, Apium graveolens and Coleus blumei. This was accomplished by soaking stems of these plants in the paramagnetic nitroxide imaging agent 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl. The experiments were accomplished at L-band frequency (1.4 to 1.9 gigahertz) with single-turn, flat-loop surface coils. One-dimensional imaging spectra were diagnostic of capillary structure and long-term stability.

Mechanism of plant mediated methane emission in tropical lowland rice.

Methane (CH4) is predominantly produced in lowland rice soil, but its emission from soil to atmosphere primarily depends on passage/conduit or capillary pore spaces present in rice plants. The gas transport mechanism through aerenchyma pore spaces of rice cultivars was studied to explore the plant mediated CH4 emission. Seven rice cultivars, based on the life cycle duration (LCD), were tested in tropical eastern India. Three LCD groups were, (a) Kalinga 1 and CR Dhan 204 (LCD: 110-120 days); (b) Lalat, Pooja and CR 1014 (LCD: 130-150 days); and (c) Durga and Varshadhan (LCD: 160-170 days). Rate of CH4emission, root exudates, root oxidase activities and shoot aerenchyma pore spaces were analyzed to study the mechanism of plant mediated emission from rice. Aerenchyma pore space was quantified in the hypothesis that it regulates the CH4 transportation from soil to atmosphere. The ratio of pore space area to total space was lowest in Kalinga 1 cultivar (0.29) and highest was in Varshadhan (0.43). Significant variations in the methane emission were observed among the cultivars with an average emission rate ranged from 0.86 mg m-2 h-1 to 4.96 mg m-2 h-1. The CH4 emission rates were lowest in short duration cultivars followed by medium and long duration ones. The greenhouse gas intensity considering average CH4 emission rate per unit grain yield was also lowest (0.35) in Kalinga 1 and relatively less in short and medium duration cultivars. Root exudation was higher at panicle initiation (PI) than maximum tillering (MT) stage. Lowest exudation was noticed in (197.2 mg C plant-1 day-1) Kalinga 1 and highest in Varsadhan (231.7 mg C plant-1 day-1). So we can say, the rate of CH4 emission was controlled by aerenchyma orientation, root exudation and biomass production rate which are the key specific traits of a cultivar. Identified traits were closely associated with duration and adaptability to cultivars grown in specific ecology. Therefore, there is possibility to breed rice cultivars depending on ecology, duration and having less CH4 emission potential, which could be effectively used in greenhouse gas mitigation strategies.

What preoperative factors predict postoperative sitting pelvic position one year following total hip arthroplasty?

AIMS: The aims of this study were to measure sagittal standing and sitting lumbar-pelvic-femoral alignment in patients before and following total hip arthroplasty (THA), and to consider what preoperative factors may influence a change in postoperative pelvic position. PATIENTS AND METHODS: A total of 161 patients were considered for inclusion. Patients had a mean age of the remaining 61 years (sd 11) with a mean body mass index (BMI) of 28 kg/m2 (sd 6). Of the 161 patients, 82 were male (51%). We excluded 17 patients (11%) with spinal conditions known to affect lumbar mobility as well as the rotational axis of the spine. Standing and sitting spine-to-lower-limb radiographs were taken of the remaining 144 patients before and one year following THA. Spinopelvic alignment measurements, including sacral slope, lumbar lordosis, and pelvic incidence, were measured. These angles were used to calculate lumbar spine flexion and femoroacetabular hip flexion from a standing to sitting position. A radiographic scoring system was used to identify those patients in the series who had lumbar degenerative disc disease (DDD) and compare spinopelvic parameters between those patients with DDD (n = 38) and those who did not (n = 106). RESULTS: Following THA, patients sat with more anterior pelvic tilt (mean increased sacral slope 18° preoperatively versus 23° postoperatively; p = 0.001) and more lumbar lordosis (mean 28° preoperatively versus 35° postoperatively; p = 0.001). Preoperative change in sacral slope from standing to sitting (p = 0.03) and the absence of DDD (p = 0.001) correlated to an increased change in postoperative sitting pelvic alignment. CONCLUSION: Sitting lumbar-pelvic-femoral alignment following THA may be driven by hip arthritis and/or spinal deformity. Patients with DDD and fixed spinopelvic alignment have a predictable pelvic position one year following THA. Patients with normal spines have less predictable postoperative pelvic position, which is likely to be driven by hip stiffness. Cite this article: Bone Joint J 2018;100-B:1289-96.

Effect of monocyte migration on low density lipoprotein transport across aortic endothelial cell monolayers.

Endothelial cell monolayers on polycarbonate filters present a barrier to low density lipoprotein (LDL) and albumin transport. These cells form a relatively tight monolayer as shown by measurements of electrical resistance across the monolayer (15 omega-cm2). Monocytes are able to migrate freely across the monolayers in response to chemotactic stimuli. Monocyte chemotaxis across the monolayer caused a marked increase in LDL and albumin transport across the monolayer in the direction of monocyte migration. However, transport in the opposite direction was not significantly increased. These results suggest that monocyte migration across the endothelium could lead to an increased LDL content of the intima.

Low density lipoproteins transfer bacterial lipopolysaccharides across endothelial monolayers in a biologically active form.

Rabbit aortic endothelial cells (RAECs) were grown on micropore filters in a device that allowed in situ determination of transendothelial electrical resistance (TEER). Incubation of confluent RAEC monolayers with 2 ng.ml-1 of bacterial LPS for 3 h did not change the protein content or the number of cells on the filters, but resulted in a marked decline in TEER (from 14.1 +/- 0.9 to 5.1 +/- 0.6 omega.cm2) and a significant increase in LDL transport across the monolayers (from 154 +/- 13 to 456 +/- 41 ng. h-1 per cm2). In contrast, exposure of RAEC monolayers for 3 d to as much as 5 micrograms.ml-1 of LPS complexed to LDL (LPS-LDL) did not alter the TEER or LDL transport. LPS-LDL was transported across the monolayers at the same rate as LDL. While microgram quantities of LPS complexed to LDL did not disrupt the integrity of the endothelial monolayer, incubation of RAECs with transported LPS-LDL at concentrations of 25-100 ng LPS.ml-1 resulted in a two- to ninefold increase in the secretion of monocyte chemotactic activity by these cells. Incubation of rabbit aortic smooth muscle cells with transported LPS-LDL at concentrations of 25-100 ng LPS.ml-1 resulted in a two- to threefold increase in the secretion of monocyte chemotactic activity. We propose that LDL protects endothelial cells from the acute toxicity of LPS but the resulting complexes are transported across the endothelium in a biologically active form that can initiate an inflammatory response.

Minimally modified low density lipoprotein-induced inflammatory responses in endothelial cells are mediated by cyclic adenosine monophosphate.

We have previously shown that minimally oxidized LDL (MM-LDL) activated endothelial cells to increase their interaction with monocytes but not neutrophils, inducing monocyte but not neutrophil binding and synthesis of monocyte chemotactic protein-1 and monocyte colony-stimulating factor (M-CSF). In the present studies we have examined the signaling pathways by which this monocyte-specific response is induced. Both induction of monocyte binding and mRNA levels for M-CSF by MM-LDL were not inhibited in protein kinase C-depleted endothelial cells. A number of our studies indicate that cAMP is the second messenger for the effects of MM-LDL cited above. Incubation of endothelial cells with MM-LDL caused a 173% increase in intracellular cAMP levels. Agents which increased cAMP levels, including cholera toxin, pertussis toxin, dibutyryl cAMP, and isoproterenol mimicked the actions of MM-LDL. Agents which elevated cAMP were also shown to activate NF kappa B, suggesting a role for this transcription factor in activation of monocyte-endothelial interactions. Although endothelial leukocyte adhesion molecule (ELAM) mRNA synthesis can be regulated by NF kappa B, ELAM was not expressed and ELAM mRNA was only slightly elevated in response to MM-LDL. We present evidence that induction of neutrophil binding by LPS is actually suppressed by agents that elevated cAMP levels.

Minimally modified low density lipoprotein is biologically active in vivo in mice.

Minimally modified low density lipoprotein (MM-LDL), derived by mild iron oxidation or prolonged storage at 4 degrees C, has been shown to induce certain inflammatory responses in vascular cells in tissue culture. These include induction of monocyte (but not neutrophil) adherence to endothelial cells (EC), induction of EC production of colony stimulating factors (CSF), and induction of EC and smooth muscle cell production of monocyte chemotactic protein (MCP-1). To test for biologic activity in vivo, microgram quantities of MM-LDL were injected into mice, sera were assayed for CSF activity, and tissues were subjected to Northern analysis. After injection of MM-LDL, CSF activity increased approximately 7-26-fold but remained near control levels after injection of native LDL. Essentially all of the induced CSF activity was due to macrophage CSF as judged by antibody inhibition. Injection of MM-LDL into a mouse strain (C3H/HeJ) that is resistant to bacterial LPS gave similar results, indicating that the induction of CSF was not due to contaminating LPS and suggesting that there are differences in the pathways by which LPS and MM-LDL trigger cytokine production. In addition, after injection of MM-LDL, mRNA for JE, the mouse homologue of MCP-1, was markedly induced in various tissues, but was not induced after injection of native LDL. We conclude, therefore, that MM-LDL is biologically active in vivo and may contribute to the early stages of atherosclerosis by acting as an inflammatory agent.

Minimally modified low density lipoprotein stimulates monocyte endothelial interactions.

The effect of minimally modified LDL (MM-LDL) on the ability of large vessel endothelial cells (EC) to interact with monocytes and neutrophils was examined. These LDL preparations, obtained by storage or by mild iron oxidation, were indistinguishable from native LDL to the LDL receptor and were not recognized by the scavenger receptor. Treatment of EC with as little as 0.12 micrograms/ml MM-LDL caused a significant increase in the production of chemotactic factor for monocytes (sevenfold) and increased monocyte binding (three- to fivefold). Monocyte binding was maximal after 4 h of EC exposure to MM-LDL, persisted for 48 h, and was inhibited by cycloheximide. In contrast, neutrophil binding was not increased after 1-24 h of exposure. Activity in the MM-LDL preparations was found primarily in the polar lipid fraction. MM-LDL was toxic for EC from one rabbit but not toxic for the cells from another rabbit or any human umbilical vein EC. The resistant cells became sensitive when incubated with lipoprotein in the presence of cycloheximide, whereas the sensitive strain became resistant when preincubated with sublethal concentrations of MM-LDL. We conclude that exposure of EC to sublethal levels of MM-LDL enhances monocyte endothelial interactions and induces resistance to the toxic effects of MM-LDL.

Monocyte transmigration induced by modification of low density lipoprotein in cocultures of human aortic wall cells is due to induction of monocyte chemotactic protein 1 synthesis and is abolished by high density lipoprotein.

Incubation of cocultures of human aortic endothelial (HAEC) and smooth muscle cells (HASMC) with LDL in the presence of 5-10% human serum resulted in a 7.2-fold induction of mRNA for monocyte chemotactic protein 1 (MCP-1), a 2.5-fold increase in the levels of MCP-1 protein in the coculture supernatants, and a 7.1-fold increase in the transmigration of monocytes into the subendothelial space of the cocultures. Monocyte migration was inhibited by 91% by antibody to MCP-1. Media collected from the cocultures that had been incubated with LDL induced target endothelial cells (EC) to bind monocyte but not neutrophil-like cells. Media collected from cocultures that had been incubated with LDL-induced monocyte migration into the subendothelial space of other cocultures that had not been exposed to LDL. In contrast, media from separate cultures of EC or smooth muscle cells (SMC) containing equal number of EC or SMC compared to coculture and incubated with the same LDL did not induce monocyte migration when incubated with the target cocultures. High density lipoprotein HDL, when presented to cocultures together with LDL, reduced the increased monocyte transmigration by 91%. Virtually all of the HDL-mediated inhibition was accounted for by the HDL2 subfraction. HDL3 was essentially without effect. Apolipoprotein AI was also ineffective in preventing monocyte transmigration while phosphatidylcholine liposomes were as effective as HDL2 suggesting that lipid components of HDL2 may have been responsible for its action. Preincubating LDL with beta-carotene or with alpha-tocopherol did not reduce monocyte migration. However, pretreatment of LDL with probucol or pretreatment of the cocultures with probucol, beta-carotene, or alpha-tocopherol before the addition of LDL prevented the LDL-induced monocyte transmigration. Addition of HDL or probucol to LDL after the exposure to cocultures did not prevent the modified LDL from inducing monocyte transmigration in fresh cocultures. We conclude that cocultures of human aortic cells can modify LDL even in the presence of serum, resulting in the induction of MCP-1, and that HDL and antioxidants prevent the LDL induced monocyte transmigration.

Role of the GRO family of chemokines in monocyte adhesion to MM-LDL-stimulated endothelium.

We have previously shown that treatment of endothelial cells with minimally modified LDL (MM-LDL) induces the binding of monocytes to unknown endothelial receptor molecules. We now report that a member of the GRO family of chemokines plays a role in MM-LDL-induced monocyte binding. A cDNA library made from rabbit aortic endothelial cells (RAEC) treated with MM-LDL was expression screened for molecules inducing binding of a human monocyte cell line (THP-1). A cDNA was isolated with 75% homology to GRO. GRO mRNA levels were significantly elevated after exposure of RAEC or human aortic endothelial cells (HAEC) to MM-LDL. HAEC treated with MM-LDL displayed an increase in a surface-associated protein that bound to antibody against GRO despite low levels of GRO in the medium. Antibody to GRO significantly inhibited the binding of monocytes to MM-LDL-treated RAEC and HAEC. The increase in GRO expression and monocyte binding were reduced by incubating MM-LDL-treated endothelial cells with heparin (in a method that releases heparan sulfate bound molecules from the cell surface). These results suggest that GRO related chemokines are bound to the surface of MM-LDL-treated endothelial cells and may contribute to the monocyte adhesion induced by MM-LDL.

Recipient mononuclear cell recognition and adhesion to graft endothelium after human cardiac transplantation. Lymphocyte recognition leads to monocyte adhesion.

Transendothelial migration of mononuclear cells is crucial in the development of allograft rejection and transplant coronary disease. Adhesion of circulating cells to endothelium is the initial step in transendothelial migration. Human aortic endothelial cell cultures were established from aortic tissue harvested at the time of organ donation for cardiac transplantation which allowed specific recipient mononuclear cell-graft endothelial interactions to be studied. Confluent untreated endothelial cells were incubated with recipient mononuclear cells for 15 min to assess adhesion. Adhesion of recipient mononuclear cells to endothelium derived from their graft was threefold higher than adhesion to nonspecific endothelium (93 +/- 20 vs. 30 +/- 11 cells/high power field, P < 0.005). Graft-specific adhesion was inhibited by preincubation of the endothelium with antibodies to class I HLA (34 +/- 16 cells/high power field, P < 0.005). Immunofluorescence performed after adhesion showed that 73 +/- 6% of both specific and nonspecific adherent cells were monocytes. The use of purified lymphocyte and monocyte preparations showed that graft-specific lymphocytes induce unrelated monocytes to become adherent. These results suggest that lymphocytes are primed in vivo to recognize endothelium derived from their graft which leads to a rapid increase in lymphocyte and monocyte adhesion. Such allo-recognition may involve endothelial class I HLA molecules.

Rabbit beta-migrating very low density lipoprotein increases endothelial macromolecular transport without altering electrical resistance.

Rabbit aortic endothelial cells (RAEC) were grown on micropore filters in a new device. This system allowed in situ measurement of transendothelial electrical resistance (TEER). The monolayers demonstrated a TEER of 14 +/- 1 omega X cm2 at confluence. No difference was seen in the transport of low density lipoproteins (LDL) across endothelial cell monolayers obtained from normal or Watanabe heritable hyperlipidemic rabbits, indicating that the LDL receptor was not involved in the LDL transport. TEER was inversely correlated with 22Na transport (r2 = 0.93, P = less than 0.001) but not with 125I-LDL transport. The amount of LDL transported at 15 degrees C or across glutaraldehyde-fixed monolayers was half that of the controls at 37 degrees C. Preincubation of the monolayers with rabbit beta-migrating very low density lipoproteins (beta-VLDL) increased cholesterol content by 65%, and the transport of albumin and LDL doubled without a change in TEER. Removal of beta-VLDL from the culture medium resulted in the return of cellular cholesterol content and LDL transport to control values. We conclude that preincubation of RAEC with beta-VLDL resulted in an increased permeability to LDL and albumin, and that beta-VLDL may promote increased transendothelial transport of macromolecules in cholesterol-fed rabbits.