Effect of 12-O-tetradecanoylphorbol-13-acetate-induced psoriasis-like skin lesions on systemic inflammation and atherosclerosis in hypercholesterolaemic apolipoprotein E deficient mice.

BACKGROUND: Risk of cardiovascular disease is increased in patients with psoriasis, but molecular mechanisms linking the two conditions have not been clearly established. Lack of appropriate animal models has hampered generation of new knowledge in this area of research and we therefore sought to develop an animal model with combined atherosclerosis and psoriasis-like skin inflammation. METHODS: Topical 12-O-tetradecanoylphorbol-13-acetate (TPA) was applied to the ears twice per week for 8 weeks in atherosclerosis-prone apolipoprotein E deficient (ApoE(-/-)) mice. RESULTS: TPA led to localized skin inflammation with increased epidermal thickness, infiltration of inflammatory-like cells and augmented tissue interleukin-17F levels. Systemic effects of the topical application of TPA were demonstrated by increased plasma concentration of serum amyloid A and splenic immune modulation, respectively. However, atherosclerotic plaque area and composition, and mRNA levels of several inflammatory genes in the aortic wall were not significantly affected by TPA-induced skin inflammation. CONCLUSIONS: TPA-induced psoriasis-like skin inflammation in atherosclerosis-prone ApoE(-/-) mice evoked systemic immune-inflammatory effects, but did not affect atherogenesis. The results may question the role of psoriasis-induced inflammation in the pathogenesis of atherosclerosis in psoriasis patients.

Opposing effects of apolipoprotein m on catabolism of apolipoprotein B-containing lipoproteins and atherosclerosis.

RATIONALE: Plasma apolipoprotein (apo)M is mainly associated with high-density lipoprotein (HDL). HDL-bound apoM is antiatherogenic in vitro. However, plasma apoM is not associated with coronary heart disease in humans, perhaps because of a positive correlation with plasma low-density lipoprotein (LDL). OBJECTIVE: We explored putative links between apoM and very-low-density (VLDL)/LDL metabolism and the antiatherogenic potential of apoM in vivo. METHODS AND RESULTS: Plasma apoM was increased approximately 2.1 and approximately 1.5 fold in mice lacking LDL receptors (Ldlr(-/-)) and expressing dysfunctional LDL receptor-related protein 1 (Lrp1(n2/n2)), respectively, but was unaffected in apoE-deficient (ApoE(-/-)) mice. Thus, pathways controlling catabolism of VLDL and LDL affect plasma apoM. Overexpression (approximately 10-fold) of human apoM increased (50% to 70%) and apoM deficiency decreased ( approximately 25%) plasma VLDL/LDL cholesterol in Ldlr(-/-) mice, whereas apoM did not affect plasma VLDL/LDL in mice with intact LDL receptors. Moreover, plasma clearance of apoM-enriched VLDL/LDL was slower than that of control VLDL/LDL in mice lacking functional LDL receptors and LRP1, suggesting that apoM impairs the catabolism of VLDL/LDL that occurs independently of the LDL receptor and LRP1. ApoM overexpression decreased atherosclerosis in ApoE(-/-) (60%) and cholate/cholesterol-fed wild-type mice (70%). However, in Ldlr(-/-) mice the antiatherogenic effect of apoM was attenuated by its VLDL/LDL-raising effect. CONCLUSION: The data suggest that defect LDL receptor function leads to increased plasma apoM concentrations, which in turn, impairs the removal of VLDL/LDL from plasma. This mechanism opposes the otherwise antiatherogenic effect of apoM.

Expression of apolipoprotein B in the kidney attenuates renal lipid accumulation.

The ability to produce apolipoprotein (apo) B-containing lipoproteins enables hepatocytes, enterocytes, and cardiomyocytes to export triglycerides. In this study, we examined secretion of apoB-containing lipoproteins from mouse kidney and its putative impact on triglyceride accumulation in the tubular epithelium. Mouse kidney expressed both the apoB and microsomal triglyceride transfer protein genes, which permit lipoprotein formation. To examine de novo lipoprotein secretion, kidneys from human apoB-transgenic mice were minced and placed in medium with (35)S-amino acids. Upon sucrose gradient ultracentrifugation of the labeled medium, fractions were analyzed by apoB immunoprecipitation. (35)S-Labeled apoB100 was recovered in approximately 1.03-1.04 g/ml lipoproteins (i.e. similar to the density of plasma low density lipoproteins). Immunohistochemistry of kidney sections suggested that apoB mainly is produced by tubular epithelial cells. ApoB expression in the kidney cortex was reduced approximately 90% in vivo by treating wild type mice with apoB-antisense locked nucleic acid oligonucleotide. Inhibition of apoB expression increased fasting-induced triglyceride accumulation in the kidney cortex by 20-25% (p = 0.008). Cholesterol stores were unaffected. Treatment with control oligonucleotides with 1 or 4 mismatching base pairs affected neither the triglyceride nor the cholesterol content of the kidney cortex. The results suggest that mammalian kidney secretes apoB100-containing lipoproteins. One biological effect may be to dampen excess storage of triglycerides in proximal tubule cells.

Effects of apolipoprotein M in uremic atherosclerosis.

BACKGROUND AND AIMS: Chronic kidney disease is characterized by uremia and causes premature death, partly due to accelerated atherosclerosis. Apolipoprotein (apo) M is a plasma carrier protein for the lipid sphingosine-1-phosphate (S1P). The Apom-S1P complex associates with HDL, and may contribute to its anti-atherosclerotic effects. The role of Apom/S1P in atherosclerosis is presently controversial and has not been explored in a uremic setting. We aimed to explore whether plasma concentrations of Apom/S1P are altered by uremia and whether Apom overexpression or deficiency affects classical and uremic atherosclerosis. METHODS: Mild to moderate uremia was induced by subtotal nephrectomy (NX) in 86-92 Apoe-deficient mice that were either Apom-wild type, Apom-deficient, or overexpressed Apom (∼10 fold). The effects of uremia on plasma Apom/S1P and atherosclerosis were evaluated and compared to non-nephrectomized controls. RESULTS: Uremia increased plasma Apom by ∼25%, but not S1P. Plasma S1P was elevated by ∼300% in mice overexpressing Apom, and decreased by ∼25% in Apom-deficient mice. Apom overexpression augmented aortic root atherosclerosis and plasma cholesterol. In contrast, aortic arch atherosclerosis was unaffected by the Apom genotype. There was no effect of Apom-deficiency or Apom overexpression on uremic atherosclerosis. CONCLUSIONS: This study highlights the complexity of Apom/S1P in atherosclerosis and challenges the notion that the Apom/S1P complex is anti-atherogenic, at least in Apoe-deficient mice.

Uremia modulates the phenotype of aortic smooth muscle cells.

BACKGROUND AND AIMS: Chronic kidney disease leads to uremia and markedly accelerates atherosclerosis. Phenotypic modulation of smooth muscle cells (SMCs) in the arterial media plays a key role in accelerating atherogenesis. The aim of this study was to investigate whether uremia per se modulates the phenotype of aortic SMCs in vivo. METHODS: Moderate uremia was induced by 5/6 nephrectomy in apolipoprotein E knockout (ApoE-/-) and wildtype C57Bl/6 mice. Plasma analysis, gene expression, histology, and myography were used to determine uremia-mediated changes in the arterial wall. RESULTS: Induction of moderate uremia in ApoE-/- mice increased atherosclerosis in the aortic arch en face 1.6 fold (p = 0.04) and induced systemic inflammation. Based on histological analyses of aortic root sections, uremia increased the medial area, while there was no difference in the content of elastic fibers or collagen in the aortic media. In the aortic arch, mRNA and miRNA expression patterns were consistent with a uremia-mediated phenotypic modulation of SMCs; e.g. downregulation of myocardin, α-smooth muscle actin, and transgelin; and upregulation of miR146a. Notably, these expression patterns were observed after acute (2 weeks) and chronic (19 and 30 weeks) uremia, both under normo- and hypercholesterolemic settings. Functionally, aortic constriction was decreased in uremic as compared to non-uremic aorta segments, as measured by myography. CONCLUSIONS: Uremia modulates the phenotype of aortic SMCs as determined by mRNA/miRNA expression, an increased medial area, and decreased aortic contractility. We propose that this phenotypic modulation of SMCs precedes the acceleration of atherosclerosis observed in uremic mice.

Laser capture microdissection-based in vivo genomic profiling of wound keratinocytes identifies similarities and differences to squamous cell carcinoma.

Keratinocytes undergo a dramatic phenotypic conversion during reepithelialization of skin wounds to become hyperproliferative, migratory, and invasive. This transient healing response phenotypically resembles malignant transformation of keratinocytes during squamous cell carcinoma progression. Here we present the first analysis of global changes in keratinocyte gene expression during skin wound healing in vivo, and compare these changes to changes in gene expression during malignant conversion of keratinized epithelium. Laser capture microdissection was used to isolate RNA from wound keratinocytes from incisional mouse skin wounds and adjacent normal skin keratinocytes. Changes in gene expression were determined by comparative cDNA array analyses, and the approach was validated by in situ hybridization. The analyses identified 48 candidate genes not previously associated with wound reepithelialization. Furthermore, the analyses revealed that the phenotypic resemblance of wound keratinocytes to squamous cell carcinoma is mimicked at the level of gene expression, but notable differences between the two tissue-remodeling processes were also observed. The combination of laser capture microdissection and cDNA array analysis provides a powerful new tool to unravel the complex changes in gene expression that underlie physiological and pathological remodeling of keratinized epithelium.

Extracellular protease mRNAs are predominantly expressed in the stromal areas of microdissected mouse breast carcinomas.

Solid tumors synthesize a number of extracellular matrix-degrading proteases that are important for tumor progression. Based on qualitative in situ hybridization studies in human cancer tissue, a range of components involved in proteolysis appear to be expressed by stromal cells rather than cancer cells. We have now used laser capture microdissection and real-time PCR to quantify the mRNA expression of components of matrix-degrading proteolytic systems in cancer and stromal areas of mouse mammary tumors genetically induced by the polyoma virus middle T (PyMT) antigen. We examined the mRNA levels of urokinase plasminogen activator, plasminogen activator inhibitor 1 and the matrix metalloproteases MMP-2, -3, -11, -13 and -14, and found that all these seven genes are predominantly expressed by stromal cells. Our results were qualitatively supported by in situ hybridization analysis of the expression of mRNAs for MMP-2, -3 and -13 in the PyMT tumors. Statistical analyses indicated that the quantitative expression patterns observed in cancer and stromal cells isolated from individual tumors from different PyMT mice are quite reproducible. The methodology described in this study provides excellent tools to study the possible interactions between cancer and stromal cells during the development of breast cancer, and the results suggest that stromal cells are involved in carcinogenesis and tumor progression, which may have important implications for the biology and therapy of cancer.