CD36 and lipid metabolism in the evolution of atherosclerosis.

Background: CD36 is a multi-functional class B scavenger receptor, which acts as an important modulator of lipid homeostasis and immune responses. Sources of data: This review uses academic articles. Areas of agreement: CD36 is closely related to the development and progression of atherosclerosis. Areas of controversy: Both persistent up-regulation of CD36 and deficiency of CD36 increase the risk for atherosclerosis. Abnormally up-regulated CD36 promotes inflammation, foam cell formation, endothelial apoptosis, macrophage trapping and thrombosis. However, CD36 deficiency also causes dyslipidemia, subclinical inflammation and metabolic disorders, which are established risk factors for atherosclerosis. Growing points: There may be an 'optimal protective window' of CD36 expression. Areas timely for developing research: In addition to traditionally modulating protein functions using gene overexpression or deficiency, the modulation of CD36 function at post-translational levels has recently been suggested to be a potential therapeutic strategy.

The PPARalpha ligand fenofibrate: meeting multiple targets in diabetic nephropathy.

Fibrate peroxisome proliferator-activated receptor (PPAR)-alpha ligands are mainly used as hypolipidemic drugs. But this commentary highlights their potential in treating insulin resistance, dyslipidemia, and hypertension and in preventing diabetic nephropathy, inflammation, and cardiovascular disease. Because diabetes is a major contributor to chronic kidney disease and cardiovascular disease, PPAR-alpha agonists may provide greater opportunities for hitting multiple targets in this complex metabolic disease.

Renovascular disease is associated with low producer genotypes of the anti-inflammatory cytokine interleukin-10.

Cytokines are important mediators of inflammatory and proliferative responses in disease states including atherosclerosis. Genetic variations in cytokine production could potentially influence the outcome of these responses. The aim of this study was to determine whether cytokine gene polymorphism might influence the development of atherosclerotic renal artery stenosis. Sixty-six patients with atherosclerotic renal artery stenosis and 100 normal healthy individuals were genotyped for interleukin-10 (IL-10), tumor necrosis factor-alpha (TNF-alpha), IL-6, and IL-2 promoter region polymorphism. TNF-a, TNF-d, and IL-10 microsatellite polymorphisms were also analyzed. The frequency of the anti-inflammatory cytokine IL-10 promoter (-1082 A positive) GA and AA genotypes which are associated with low production were higher in the patient group when compared to the control group. The AA-TT-AA homozygous genotype combination of three single-nucleotide polymorphisms at -1082, -819, and -592 in the IL-10 gene was also observed at a higher frequency in the patient group compared to the controls. The frequency of TNF-alpha, IL-6, and IL-2 polymorphisms did not show any significant difference between the patient and control groups. To correlate IL-10 genotypes with differences in IL-10 protein expression, in vitro mRNA and protein levels were analyzed in lipopolysaccharide-stimulated peripheral blood mononuclear cells from 22 patients with renal artery stenosis and 33 controls. Individuals genotyped as A positive at position -1082 produced lower levels of IL-10 protein and had lower copy numbers of mRNA when compared to individuals genotyped as A negative in both patient and control groups. The increased frequency of the low producer IL-10 promoter, -1082 A-positive genotype in patients with renal artery stenosis, suggests that IL-10 may protect against the development of atherosclerotic renovascular disease.

Nitric oxide production by human peritoneal mesothelial cells.

Nitric oxide (NO) has multiple actions, ranging from immunomodulation to regulation of vascular tone and capillary flow. Thus NO generation within the peritoneum could potentially affect peritoneal transport by increasing capillary vasodilatation, and regulate the response to bacterial invasion. Peritoneal mesothelial cells have a common embryological derivation with endothelial cells. As mesothelial cells are the predominant cell type lining the peritoneal cavity, they could potentially be a major source of locally produced nitric oxide. Nitric oxide was measured using the Griess reaction, as total nitrite and nitrate, in fresh unused and spent dialysate effluent (SPDE) from both healthy peritoneal dialysis patients, and during episodes of bacterial peritonitis. Whereas fresh CAPD dialysate was nitrite free (5 +/- 0.1 microM), SPDE from a standard 4 h day time exchange contained 10.2 +/- 0.6 microM/L/h, and that from the overnight dwell 9.1 +/- 0.7 microM/L/h. During an episode of peritonitis, dialysate nitrite and nitrate increased significantly from 9.0 +/- 1.0 microM/L/h, when not infected to 17.5 +/- 2.4, from the first CAPD bag drained at presentation, and 15.2 +/- 1.8 for the second and 16.0 +/- 2.5 for the third exchange (p<0.01). By the following day nitrite levels had returned to baseline, 7.0 +/- 1.0 microM/L/h. Human peritoneal mesothelial cells (HPMC) were cultured and found to produce nitric oxide (261 nmol/mg cell protein), which increased in a dose dependent manner with the addition of spent uninfected CAPD dialysate. The addition of L-arginine, a NO substrate resulted in a 10% increase in nitric oxide production, whereas the addition of the blocker L-NMMA produced a 10% reduction. RNA for inducible nitric oxide synthase (iNOS) was sought using northern blotting technique following combination stimulation with lipopolysaccharide and cytokines (IL-1beta, TNFalpha and gamma-INF, and/or spent dialysate from patients with bacterial peritonitis). However, we could not demonstrate RNA production for iNOS. Peritoneal mesothelial cells may be an important source of locally generated nitric oxide within the peritoneal cavity under basal conditions, but as they do not contain iNOS, the markedly increased NO production observed with episodes of acute bacterial peritonitis is more likely due to a combination of increased NO production by peritoneal macrophages and endothelial cells.

Regulation of lipoprotein trafficking in the kidney: role of inflammatory mediators and transcription factors.

Inflammation and dyslipidaemia both play important roles in the development of glomerular atherosclerosis in renal diseases. We have demonstrated that inflammatory mediators induced Scr (scavenger receptor) expression and the formation of foam cells, and that AP-1 (activator protein 1)/ets were necessary transcriptional factors for Scr induction in HMCs (human kidney mesangial cells). Most cells are protected from excessive native LDL (low-density lipoprotein) accumulation by tight feedback regulation of the LDLr (LDL receptor). However, we observed that HMCs formed foam cells via the LDLr pathway when incubated with IL-1beta (interleukin-1beta; 5 ng/ml) and unmodified LDL (200 microg/ml), suggesting that inflammatory mediators may disrupt the cholesterol-mediated feedback regulation. This feedback involves cholesterol-mediated down-regulation of LDLr controlled by SCAP [SREBP (sterol responsive element-binding protein) cleavage-activating protein]. We have also demonstrated that both tumour necrosis factor alpha and IL-1beta increased nuclear SREBP-1 levels by increasing SCAP mRNA expression, even in the presence of a high concentration of LDL. Since intracellular lipid content is governed by both influx and efflux mechanisms, we set out to examine the impact of inflammatory cytokines on cholesterol efflux, a process mediated by the protein ABCA1 (ATP binding cassette A1). IL-1beta inhibited [(3)H]cholesterol efflux from HMCs by inhibition of the peroxisome-proliferator-activated receptor/LXR (liver X receptor)/ABCA1 pathway. Taken together, our results suggest that inflammatory mediators increase lipid accumulation in HMCs not only by promoting increased lipoprotein uptake by Scr and LDLr, but also by inhibiting ABCA1-mediated cholesterol efflux to high-density lipoprotein.

Does oral folic acid lower total homocysteine levels and improve endothelial function in children with chronic renal failure?

BACKGROUND: Accelerated vascular disease is common in chronic renal failure (CRF) and accounts for significant mortality and morbidity. Elevated homocysteine levels may contribute by an effect on endothelial function. METHODS AND RESULTS: We performed a double-blind placebo-controlled randomized crossover trial of folic acid at 5 mg/m2 in 25 normotensive children 12+/-3 (7 to 17) years of age with CRF (glomerular filtration rate 26.8+/-13.2 mL/min per 1.73 m2) of noninflammatory etiology. Each subject underwent two 8-week periods of folic acid and placebo separated by an 8-week washout period. The effect of folic acid on homocysteine levels, LDL oxidation, and both endothelial-dependent and -independent vascular function were measured. After oral folic acid, serum folate levels rose from 11.7+/-4.25 to 635+/-519 microg/L (P=0.001), red cell folate levels rose from 364+/-195 to 2891+/-2623 microg/L (P<0.001), and total homocysteine levels fell from 10.28+/-4.16 to 8.62+/-2.32 micromol/L (P=0.03). In addition, there was a significant improvement in flow-mediated dilatation (FMD) (endothelial-dependent dilatation) from 7.21+/-2.8% to 8.47+/-3.01% (P=0.036) with no change in response to glyceryl trinitrate (endothelial-independent dilatation). There was no significant change in FMD or glyceryl trinitrate during the placebo phase. There was, however, no significant difference in final FMD after placebo or folic acid. Lag times for LDL oxidation were prolonged during the treatment phase (58.4+/-18.7 to 68.1+/-25.9 minutes, P=0.01). CONCLUSION: Folic acid supplementation in children with CRF may improve endothelial function with an increased resistance of LDL to oxidation.

Dysregulation of LDL receptor under the influence of inflammatory cytokines: a new pathway for foam cell formation.

BACKGROUND: Lipid-mediated renal injury is an important component of glomerulosclerosis and its similarity to atherosclerosis is well described. This study focused on the relationship between lipid-mediated injury and inflammation by examining the role of inflammatory cytokines in the regulation of human mesangial cell low-density lipoprotein (LDL) receptors. METHODS: A human mesangial cell line (HMCL) was used to study the effects of tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) on the regulation of LDL receptor mRNA and protein in the presence of a high concentration of native LDL (250 microg/mL). RESULTS: Native LDL caused foam cell formation in HMCL in the presence of antioxidants, TNF-alpha and IL-1beta. Both cytokines overrode LDL receptor suppression induced by a high concentration of LDL and increased LDL uptake by enhancing receptor expression. These cytokines also caused increased expression of SCAP [sterol responsive element binding protein (SREBP) cleavage activation protein], and an increase in the nuclear translocation of SREBP, which induces LDL receptor expression. CONCLUSION: These observations demonstrate that inflammatory cytokines can modify cholesterol-mediated LDL receptor regulation in mesangial cells, permitting unregulated intracellular accumulation of unmodified LDL and causing foam cell formation. These findings suggest that inflammatory cytokines contribute to lipid-mediated renal damage, and also may have wider implications for the study of inflammation in the atherosclerotic process.

Paradoxical increase in nitric oxide synthase activity in hypercholesterolaemic rats with impaired renal function and decreased activity of nitric oxide.

BACKGROUND: We have shown that acute exposure of oxidized low-density lipoprotein (OX-LDL) induces vasoconstriction in renal vessels and reduces glomerular filtration rate (GFR) in an isolated perfused rat kidney model by decreasing the activity of nitric oxide (NO). L-arginine has a protective role against OX-LDl-induced vasoconstriction. Micropuncture studies have demonstrated that short-term diet-induced hypercholesterolaemia is associated with decreased GFR and renal blood flow and increased glomerular capillary pressure. This may be mediated by decreased activity of NO. METHODS: Rats were made hypercholesterolaemic by supplementing the standard chow with 4% cholesterol and 1% sodium cholate. A group of rats on hypercholesterolaemic diet also received L-arginine in the drinking water. After 4 and 6 weeks, blood samples and 24-h urine samples were collected for the measurement of biochemical parameters. After 6 weeks, all rats were subjected to isolated perfusion of kidneys at a constant pressure of 100 mmHg. During isolated perfusion, the unused contralateral kidney was taken for morphological studies and for assessing the activity of nitric oxide synthase enzyme by beta-NADPH diaphorase histochemistry. RESULTS: Rats fed a high-cholesterol diet had LDL levels 3-6 times greater than the rats fed standard chow. Rats that received L-arginine in the drinking water had serum L-arginine levels 5-6 times greater than control rats. At 6 weeks, creatinine clearance was significantly lower in the rats on the high-cholesterol diet compared to the rats on standard chow and rats on high-cholesterol diet plus L-arginine. Twenty-four-hour urinary total nitrate and nitrite excretion in the hypercholesterolaemic rats was 1.5-2 times greater than that of control rats. Twenty-four-hour urinary cGMP excretion was significantly lower in the rats on a high-cholesterol diet, but in the rats on high-cholesterol diet and L-arginine, 24-h urinary cGMP excretion was not significantly different from that of control rats. During isolated perfusion of kidneys, renal perfusate flow was found to be significantly reduced in the kidneys taken from the rats on a high-fat diet compared to controls. L-arginine supplementation in the drinking water almost completely reversed the effect of a high-fat diet. Inulin clearance was also significantly reduced in kidneys on a high-fat diet in contrast to controls but not in kidneys on high fat-diet and L-arginine. Basal cGMP excretion in urine was significantly lower in the kidneys taken from the rats on a high-fat diet compared to controls. L-arginine supplementation restored the basal cGMP excretion in these kidneys. NO synthase (NOS) enzyme activity as assessed by NADPH diaphorase activity showed that kidney sections taken from the rats on a high-fat diet showed more intense staining, indicating increased activity compared to the kidney sections taken from the rats on a normal diet. CONCLUSION: Though activity of NO is diminished in hypercholesterolaemic rats with impaired renal function, there is a paradoxical increase in NO production and NOS activity. L-arginine reverses the effects of a high-fat diet.

Human mesangial cells express inducible macrophage scavenger receptor.

BACKGROUND: Type A scavenger receptors (Scr) mediate the uptake of modified low-density lipoproteins by macrophages. The accumulation of lipids via this process is thought to lead to foam cell formation in atherosclerotic plaques. Human mesangial cells (HMCs) have not been previously shown to express Scr in normal culture. We therefore investigated whether there is an inducible form of Scr in a human mesangial cell line (HMCL). METHODS: Scr activity was analyzed by cellular uptake of fluorescently labeled acetylated low-density lipoprotein using a flow cytometer. Scr mRNA expression was examined using reverse transcription-polymerase chain reaction, followed by Southern blotting. To investigate the molecular mechanism of Scr expression, several reporter gene constructs were designed. The first contained a full Scr promoter, the second a part of the Scr promoter that has both AP-1 and ets transcription factor binding sites. Other constructs were identical to the second, except that they contained either AP-1 or ets motif mutations. RESULTS: Phorbol 12-Myristate 13-acetate (PMA) and angiotensin II (Ang II) increased both the percentage of Scr-positive cells and the Scr mean fluorescence intensity. PMA and Ang II also increased Scr mRNA and promoter activity in a time- and dose-responsive manner. Protein kinase C and calmodulin transduction pathways were involved in Scr up-regulation induced by PMA and Ang II. Additionally, a serine/threonine kinase was involved in PMA stimulation. Functional analysis showed that both AP-1 and ets motifs were specific response elements to PMA stimulation in HMCLs. CONCLUSIONS: This study suggests that HMCs may express an inducible Scr, by which cells can acquire lipids and convert to foam cells in developing glomerulosclerosis.

Lysophosphatidylcholine induces platelet-derived growth factor gene expression in a human mesangial cell line.

BACKGROUND: Oxidized low-density lipoprotein (oxLDL) has been considered important in the pathogenesis of progressive renal injury. Lysophosphatidylcholine (lysoPC) is a major phospholipid component of oxLDL. On the other hand, platelet-derived growth factor (PDGF) has also been implicated in proliferative disease of the kidney. This study investigated the difference in the potential of PC and lysoPC to induce DNA synthesis and PDGF gene expression in a human glomerular mesangial cell line (HMCL). METHODS: DNA synthesis in HMCL was measured by [3H] thymidine incorporation. The mRNA expression levels of the PDGF A chain and B chain genes were measured using reverse transcription-polymerase chain reaction. RESULTS: LysoPC treatment up-regulated the [3H] thymidine incorporation level in a dose-dependent fashion. The [3H] thymidine incorporation level in HMCL coincubated with lysoPC started to increase after 4 hours of treatment, peaked at 24 hours, and decreased thereafter. The level in HMCL incubated with 100 microM of lysoPC (palmitoyl or stearoyl) increased to 7- or 10-fold of the control at peak time, respectively. However, PC treatment did not increase [3H] thymidine incorporation in HMCL. PC treatment did not induce mRNA expression of either PDGF A or B chain genes. LysoPC did not induce PDGF A chain mRNA expression either. The only B chain mRNA expression was induced by lysoPC. The mRNA expression level in HMCL treated with 50 microM lysoPC for two hours increased to 1.6-fold that of the control. CONCLUSION: LysoPC may induce DNA synthesis in a mesangial cell through the induction of PDGF BB as an autocrine and paracrine growth factor.

Lipopheresis in the nephrotic syndrome.

BACKGROUND: Experimental models have established a role for lipoproteins in the pathogenesis of progressive renal failure. However, conventional treatment rarely normalizes the high serum cholesterol of the nephrotic syndrome. The removal of low-density lipoprotein by lipopheresis is discussed. METHODS: Lipopheresis may be beneficial in nephrotic patients with focal segmental glomerulosclerosis. The authors studied the long-term effects of low-density lipoprotein cholesterol (LDL-C) removal using the Kaneka Liposorber system, which binds LDL-C to dextran sulfate in a controlled trial in 20 nephrotic patients with different renal diseases. RESULTS: A 21-month clamp of plasma total cholesterol at 6.0 mmol/liter or below was significantly lower than controls (chi 2 = 84.3, P < 0.001), followed 12 aphereses over 6 to 12 weeks in all but three apheresed patients. 1/Cr slopes were unchanged when the 50-day average period of lipopheresis treatments was excluded from analysis. Proteinuria was not reduced, but serum albumin tended to rise (NS). Fibrinogen fell by 29.8%; high-density lipoprotein, apoA1, and Lp(a) were unchanged. Two apheresed patients had a prolonged remission with a reduction of proteinuria to less than 250 mg/24 hr. The reasons for prolonged reduction of total cholesterol include depletion of tissue cholesterol, an improved fractional catabolic rate of very low density lipoprotein (VLDL), increased hepatocyte LDL turnover, and the maintenance of statin therapy. CONCLUSION: Lipopheresis is a safe and effective method for the control of LDL in nephrotic syndrome. Early clamping of total cholesterol in the normal range resulted in a prolonged and significant reduction of LDL compared with controls.

Calcineurin inhibitors enhance low-density lipoprotein oxidation in transplant patients.

BACKGROUND: Our objective was to assess the pro-oxidant status of neoral and tacrolimus in renal transplant patients and monitor the protection provided by vitamin C and vitamin E in normalizing low density lipoprotein (LDL) oxidation lag time of tacrolimus-treated patients. METHODS: Plasma LDL was isolated by density gradient ultracentrifugation from renal transplant patients receiving neoral, tacrolimus and tacrolimus with vitamin C and vitamin E. Oxidation was initiated by the addition of CuCl2 at 37 degrees C and monitored at 234 nm over 480 minutes and oxidation lag time was computed. Total antioxidant capacity of serum was measured using the enhanced chemiluminescent method. RESULTS: LDL from tacrolimus-treated patients had significantly lower oxidation lag time and serum antioxidant activity in comparison with neoral-treated patients, and this was particularly significant during the first four months after transplantation. Vitamin C and E supplementation in tacrolimus treated patients provided protection against oxidation and normalized their oxidation lag time. CONCLUSION: Calcineurin-inhibiting drugs, CsA and tacrolimus, have pro-oxidant activity and they increase the susceptibility of LDL to oxidation. Neoral formulation is fortified with DL-alpha tocopherol and therefore provides protection against oxidation. The present study clearly demonstrates the benefit of giving vitamin C and E supplements to patients taking tacrolimus and this seems to be particularly important during the early period after transplantation.

Human mesangial cells express inducible macrophage scavenger receptor: an Ap-1 and ets mediated response.

BACKGROUND: Type A scavenger (SR) mediate the uptake of modified low-density lipoproteins by macrophages. The accumulation of lipid via this process is thought to lead to foam cell formation in atherosclerotic plaques. Human mesangial cells (HMC), which can be converted to foam cells in vivo, have not previously been shown to express SR in normal culture. We investigated whether or not there was an inducible form of SR in a human mesangial cell line (HMCL). METHODS: SR activity was analyzed by cellular uptake of fluorescently labeled acetylated low-density lipoprotein using a flow cytometer. SR mRNA expression was examined using RT-PCR followed by Southern blotting. To investigate the molecular mechanism of SR expression, several reporter gene constructs were designed. The first contained a full SR promoter, the second a part of the SR promoter that has both activated protein-1 (AP-1) and ets transcriptional factor binding sites. Other constructs were identical to the second except they contained either AP-1 or ets motif mutations. RESULTS: Phorbol 12-myristate 13-acetate (PMA) increased both the percentage of SR positive cells and SR mean fluorescence intensity. PMA also increased SR mRNA and promoter activity in a time and dose responsive manner. Function analysis showed that both AP-1 and ets motifs were specific response elements to PMA stimulation in HMCL. CONCLUSIONS: The present study suggests that the combination of interaction between AP-1 and ets transcriptional factors may mediate the inducible expression of the SR gene in HMCL, which may contribute to foam cell formation.

LDL receptor gene expression in human mesangial cells under the influence of calcium channel blockers.

BACKGROUND: Intracellular transport of lipid through the regulation of LDL receptor (LDLr) may be important in the progression of renal dysfunction. METHODS: We explored LDLr gene expression in human mesangial cell line (HMCL) under influence of calcium channel blockers using cell proliferation, LDL binding, Northern blot and LDLr promoter activity assay. RESULTS: Diltiazem and verapamil increased the expression of LDLr mRNA in a dose-dependent manner. Increased LDLr mRNA paralleled LDL binding. Nifedipine did not increase the expression of LDLr mRNA and LDL binding to HMCL at 1 - 100 micromol/l. The LDLr promoter activity assay showed that treatment with 100 micromol/ of diltiazem and verapamil increased LDLr promoter activity by 126.72 +/- 10.68%, and 166.41 +/- 11.41%, respectively, at 24 hours (control as 100%), while treatment with 100 micromol/l of nifedipine had an inhibitory effect on LDLr promoter activity. High concentration of LDL (250 microg/ml) inhibited promoter activity. Diltiazem or verapamil coincubated with LDL (250 microg/ml) could not override transcriptional inhibition by LDL. CCBs inhibited the proliferation of HMCL, therefore, CCBs-induced LDLr expression did not depend on a proliferative response. Signal transduction pathway experiments showed that the calmodulin transduction pathway was involved in LDLr upregulation induced by diltiazem or verapamil. Additionally, tyrosine kinase and PKC pathways were involved in the induction of LDLr induced by verapamil. CONCLUSION: These studies show that diltiazem and verapamil increase LDLr gene transcription and expression which is independent of cell proliferation in HMCL.

Renal vasoconstriction induced by oxidized LDL is inhibited by scavengers of reactive oxygen species and L-arginine.

BACKGROUND: Low density lipoprotein (LDL) may be involved in the pathogenesis of glomerulosclerosis and progressive renal dysfunction associated with atherosclerotic renal artery stenosis (RAS). This study was undertaken to investigate the effects of native (n-LDL) and oxidized LDL (ox-LDL) on renal vascular response and function in an isolated perfused rat kidney (IPRK) model. MATERIAL AND METHOD: IPRK model was used for the study at a constant pressure of 100 mm of Hg in the renal artery with continuous monitoring of pressure and renal perfusate flow. Urine and perfusate samples were collected to determine [14C] Inulin clearance and fractional reabsorption of sodium. To elucidate the role of nitric oxide (NO) urinary c-GMP, nitrate and nitrite excretion were measured and the responses to the NO synthase inhibitor N-monomethyl-L-arginine (LNMMA) and the NO donor Nitroso-glutathione (GSNO) were assessed. The effect of L-arginine supplementation and the role of reactive oxygen species were also studied by adding superoxide dismutase (SOD) and catalase. RESULTS: Ox-LDL but not n-LDL caused vasoconstriction in IPRK, as evidenced by a significant dose dependent reduction in renal perfusate flow. [14C] Inulin clearance and fractional reabsorption of sodium were reduced during ox-LDL infusion whereas no significant change occured with n-LDL. There was a significant decrease in urinary excretion of c-GMP during ox-LDL infusion. 10 microM LNMMA significantly increased and GSNO (10 microM) significantly diminished the vasoconstrictory effect of ox-LDL. The presence of L-arginine (100 & 500 microM) significantly decreased ox-LDL induced vasoconstriction. SOD (150 U/ml) and catalase (1200 U/ml) both had a significant inhibitory effect and the combination of SOD and catalase almost completely abolished the vasoconstriction due to ox-LDL. CONCLUSION: These results suggest that ox-LDL induced vasoconstriction in IPRK is mediated by decreased activity of NO probably due to inactivation of NO by reactive oxygen species. The free radical scavengers SOD, catalase and L-arginine provided protection against ox-LDL induced vasoconstriction in this model.