Neutrophil infiltration and systemic vascular inflammation in obese women.

Obesity has become epidemic worldwide and is especially pronounced in women of reproductive age, which is important because obesity is a major risk factor for preeclampsia and chronic hypertension. We hypothesized that vascular inflammation is critical to the pathophysiology of hypertension in obese individuals because obesity and hypertensive disorders share common features related to inflammation. To study this, we collected subcutaneous fat biopsies from normal weight, overweight, and obese women and stained the tissues for CD66b, a neutrophil marker, and for activated nuclear factor-kappaB (NF-kappaB) and cyclooxygenase-2 (COX-2) as markers of inflammation. We found that the number of neutrophils per vessel and the percentage and intensity of vessel staining for CD66b, NF-kappaB and COX-2 were greatest in obese women and least in normal weight women, and that neutrophil infiltration and vascular inflammation significantly correlated with body mass index (BMI) and blood pressure. These data may help explain the relationship between obesity and hypertensive disorders.

Systemic activation and vascular infiltration of neutrophils with term labor.

OBJECTIVE: The purpose of the current study was to determine if neutrophils infiltrate maternal systemic vascular tissue at the time of term labor. METHODS: Subcutaneous fat biopsies were obtained at cesarean delivery or abdominal surgery from laboring women (n = 5), non-laboring women (n = 5), and normal non-pregnant women (n = 5). Immunohistochemical staining was performed for CD66b, a neutrophil antigen, and intercellular adhesion molecule-1 (ICAM-1; CD54), an endothelial cell adhesion molecule for neutrophils. Vessels (10 to 200 microm) were analyzed for intensity of staining and percentage of vessels with staining. RESULTS: CD66b staining intensity was significantly greater for laboring women at term than for non-laboring women at term or for normal non-pregnant women (1.3 +/- 0.3 versus 0.2 +/- 0.1 versus 0.2 +/- 0.1, respectively, P < .01). Laboring women had significantly more vessels with staining for CD66b (79 +/- 4 versus 24 +/- 8 versus 19 +/- 6%, P < .001), more vessels with neutrophils adhered and flattened to endothelium (67 +/- 3 versus 16 +/- 7 versus 12 +/- 4%, P < .001), more vessels with neutrophils in the intima (30 +/- 6 versus 5 +/- 2 versus 2 +/- 1%, P < .05), and a greater number of neutrophils per vessel (5.4 +/- 1.1 versus 1.7 +/- 0.5 versus 1.2 +/- 0.3, P < .01) as compared to non-laboring or normal non-pregnant women. ICAM-1 staining was present in the endothelium of all groups, with no difference in staining intensity or percent of vessels stained. Between 86% to 96% of vessels stained for ICAM-1. Laboring patients had numerous leukocytes stained for ICAM-1 in their vessels. CONCLUSION: This study demonstrates that neutrophils infiltrate maternal systemic vascular tissue at the time of term labor. Neutrophils were flattened and adhered to endothelium and infiltrated into the intimal space.

Modulation of adipose tissue development by pharmacological inhibition of PAI-1.

OBJECTIVE: The effect of a novel small molecule plasminogen activator inhibitor (PAI-1) inhibitor on adipose tissue physiology was investigated. METHODS AND RESULTS: In human preadipocyte cultures, PAI-039 inhibited both basal and glucose-stimulated increases in active PAI-1 antigen, yet had no effect on PAI-1 mRNA, suggesting a direct inactivation of PAI-1. Differentiation of human preadipocytes to adipocytes was associated with leptin synthesis, which was significantly reduced in the presence of PAI-039, together with an atypical adipocyte morphology characterized by a reduction in the size and number of lipid containing vesicles. In a model of diet-induced obesity, pair-fed C57 Bl/6 mice administered PAI-039 in a high-fat diet exhibited a dose-dependent reduction in body weight, epididymal adipose tissue weight, adipocyte volume, and circulating plasma active PAI-1. Plasma glucose, triglycerides, and leptin were also significantly reduced in drug-treated mice, and concentrations of PAI-039 associated with these physiological effects were near the in vitro IC50 for the inhibition of PAI-1. CONCLUSIONS: Our results indicate that a small molecule inactivator of PAI-1 can neutralize glucose-stimulated increases in PAI-1 in human preadipocyte cultures, reduce adipocyte differentiation, and prevent the development of diet-induced obesity. These data suggest the pharmacological inhibition of PAI-1 could be beneficial in diseases associated with expansion of adipose tissue mass.

Pharmacologic inhibition of platelet vWF-GPIb alpha interaction prevents coronary artery thrombosis.

Under high shear arterial blood flow von Willebrand Factor (vWF) binds the platelet receptor glycoprotein (GP) Ibalpha, leading to platelet adhesion, activation and thrombosis. Blockade of vWF-GPIb alpha interactions by GPG-290 was investigated in a canine model of coronary artery thrombosis alone and in combination with clopidogrel. GPG-290 (100 microg/kg, n=6; 500 microg/kg, n=6) prolonged time to thrombotic occlusion (TTO) to 105+/-34 and 156+/-23 (p<0.05) min, respectively compared to the saline treated control group (32+/-6 min, n=6). Patency of the injured vessel was sustained in 1/6 (100 microg/kg) and 3/6 vessels (500 microg/kg) 4 hours after injury, in contrast to 0/6 in the control group. There was an increase in bleeding after the 500 microg/kg dose, but only at the 1 hr time point. Clopidogrel was studied in two dosing regimens representing either a clinical pretreatment regimen (PTR) of 4.3 mg/kg on day -2 followed by 1.1 mg/kg daily for 2 days prior to the procedure or pre-procedural loading dose regimen (LDR) of 4.3 mg/kg 3 hr pre-procedure. The PTR and LDR clopidogrel treatments prolonged TTO to 98.2+/-30.0 min and 136.1+/-39.5 min (p<0.05), and sustained patency in 1/6 and 4/8 vessels, respectively. However, template bleeding time in the LDR clopidogrel group was sustained higher than the control group. The combination of PTR clopidogrel and GPG-290 (100 microg/kg) prolonged TTO equivalent to LDR clopidogrel alone (141.4 +/- 35.1 min) and sustained patency in 3/7 dogs, without increased bleeding while LDR clopidogrel combined with 100 microg/kg GPG-290 prevented occlusion in 5/8 dogs and further prolonged TTO (173.5+/-32.6 min) but was associated with increased bleeding compared to control. GPG-290 is an antithrombotic agent that may be combined with lower doses of clopidogrel to yield similar antithrombotic efficacy as higher loading doses.

Linoleic acid, but not oleic acid, upregulates production of interleukin-8 by human vascular smooth muscle cells via arachidonic acid metabolites under conditions of oxidative stress.

OBJECTIVE: Preeclampsia is associated with oxidative stress, elevated plasma levels of linoleic acid (LA), and increased vascular smooth muscle expression of the inflammatory chemokine, interleukin-8 (IL-8). We hypothesized that increased levels of LA under conditions of oxidative stress would increased production of IL-8 by vascular smooth muscle cells because LA is the dietary precursor to arachidonic acid (AA) and its metabolites that mediate inflammation. We also hypothesized that oleic acid (OA), which is not metabolized to AA metabolites, would not increase IL-8 under conditions of oxidative stress. METHODS: To test this hypothesis, we cultured placental arterial smooth muscle (PASM) cells with an oxidizing solution enriched with LA (OxLA) or OA (OxOA). Media concentrations were analyzed for IL-8 and AA metabolites. Inhibitors were used to block the lipoxygenase and cyclooxygenase pathways. RESULTS: Exposure of cells to OxLA, but not to OxOA, significantly increased production of IL-8. OxLA also significantly increased production of AA metabolites. Nordihydroguaiaretic acid, an inhibitor of the lipoxygenase pathway, blocked IL-8 and leukotriene B4 (LTB4) production induced by OxLA, whereas indomethacin, an inhibitor of the cyclooxygenase pathway, blocked IL-8, prostaglandin E2 (PGE2), and thromboxane B2 (TXB2) production. Reverse transcriptase-polymerase chain reaction (RT-PCR) demonstrated gene expression in PASM cells for representative lipoxygenase (LTB4) and cyclooxygenase (thromboxane) metabolite receptors. CONCLUSION: PASM cells produced IL-8 in response to LA, but not OA, under conditions of oxidative stress. The IL-8 response was mediated by AA metabolites.

Neutrophils infiltrate resistance-sized vessels of subcutaneous fat in women with preeclampsia.

We examined if there is systemic vascular inflammation and neutrophil infiltration in women with preeclampsia. Resistance-sized vessels (10 to 200 microm) of subcutaneous fat were evaluated from normal nonpregnant women, normal pregnant women, and preeclamptic women. Immunohistochemical staining was performed for: (1) interleukin-8 (IL-8), a potent neutrophil chemokine; (2) intercellular adhesion molecule-1 (ICAM-1; CD54), an endothelial cell adhesion molecule; and (3) CD66b, a neutrophil antigen. Vessels of preeclamptic patients had intense IL-8 staining in the endothelium and vascular smooth muscle, as compared with little or no staining for normal pregnant and normal nonpregnant patients. ICAM-1 was expressed on the endothelium of all patient groups. In preeclamptic patients, ICAM-1 was also expressed on vascular smooth muscle. Vessels of preeclamptic patients had significantly more CD66b staining of neutrophils than did normal pregnant or normal nonpregnant patients. There were significantly more vessels stained, more vessels with neutrophils flattened and adhered to endothelium, more vessels with neutrophils infiltrated into the intima, and more neutrophils per vessel. In conclusion, in women with preeclampsia, there was significant infiltration of neutrophils into maternal systemic vasculature associated with inflammation of the vascular smooth muscle indicated by increased expression of IL-8 and ICAM-1. Neutrophil infiltration provides a reasonable explanation for endothelial and vascular smooth muscle dysfunction in preeclampsia because neutrophils produce toxic substances, which may explain clinical symptoms.

Isolation and culture of arterial smooth muscle cells from human placenta.

A simple and economical technique was developed to isolate and culture human arterial smooth muscle cells from chorionic plate vessels. Placentas from healthy women were collected at the time of term delivery. Chorionic plate arteries were identified, excised, and cut into small pieces. An explant technique was used to grow cultures of placental arterial smooth muscle (PASM) cells. Small pieces of vessel with lumens down were placed in 100-mm culture plates and grown in Dulbecco modified eagle medium and 10% fetal bovine serum. Cells appeared from explants within 1 week and grew to confluence in approximately 4 weeks. At confluence, PASM cell cultures had a uniform cell morphology that was characterized by elongated cells in parallel rows, typical of smooth muscle cells. Smooth muscle cell phenotype was evaluated by morphology and by immunoblotting and immunofluorescence of smooth muscle myofilament proteins. All PASM cell cultures expressed alpha-smooth muscle actin, beta-tropomyosin, and h-caldesmon. Expression was similar to that of human aortic smooth muscle cells, but not to endothelial cells or fibroblasts. PASM cells stained uniformly for alpha-smooth muscle actin and lacked staining for a fibroblast-specific antigen. PASM cells were evaluated for their response to inflammatory mediators, tumor necrosis factor-alpha, and interleukin-1beta by measurement of interleukin-8 production. Cells cultured for 18 hours showed a progressive increase in interleukin-8 production with time. Treatment with inflammatory mediators increased interleukin-8 production by 3-fold as compared with media control. This technique provides a simple method to obtain normal human arterial smooth muscle cells for in vitro studies of physiology and pathophysiology.