Peroxisome proliferator-activated receptor-gamma (PPARγ) regulates the interface between cellular lipid metabolism, redox status and organelle differentiation. Conditional prostatic epithelial knockout of PPARγ in mice resulted in focal hyperplasia which developed into mouse prostatic intraepithelial neoplasia (mPIN). The grade of PIN became more severe with time. Electron microscopy (EM) showed accumulated secondary lysosomes containing cellular organelles and debris suggestive of autophagy. Consistent with this analysis the autophagy marker LC3 was found to be upregulated in areas of PIN in PPARγ KO tissues. We selectively knocked down PPARγ2 isoform in wild-type mouse prostatic epithelial cells and examined the consequences of this in a tissue recombination model. Histopathologically grafted tissues resembled the conditional PPARγ KO mouse prostates. EM studies of PPARγ- and PPARγ2-deficient epithelial cells in vitro were suggestive of autophagy, consistent with the prostatic tissue analysis. This was confirmed by examining expression of beclin-1 and LC3. Gene expression profiling in PPARγ-/γ2-deficient cells indicated a major dysregulation of cell cycle control and metabolic signaling networks related to peroxisomal and lysosomal maturation, lipid oxidation and degradation. The putative autophagic phenotypes of PPARγ-deficient cells could be rescued by re-expression of either γ1 or γ2 isoform. We conclude that disruption of PPARγ signaling results in autophagy and oxidative stress during mPIN pathogenesis.

Peroxisome proliferator-activated receptor-gamma (PPARgamma) is a major modulator of cellular lipid metabolism and organelle differentiation. To understand whether autophagy is involved in the processes of dysregulated fatty acid oxidation and induced oxidative stress accompanying prostatic carcinogenesis, we characterized in vitro and in vivo models of PPARgamma- and PPARgamma2-deficiency in mouse prostate epithelia. Autophagy accompanied the altered cellular proliferation and de-differentiation that resulted in PPARgamma-/gamma2-deficient mouse prostatic intraepithelial neoplasia (mPIN). Electron microscopy showed accumulated defective lysosomes and autophagic vacuoles in PPARgamma-/gamma2-deficient cells, suggestive of autophagy. Gene expression profiling indicated a major dysregulation of cell cycle control and metabolic signaling networks related to peroxisomal, mitochondrial and lysosomal maturation, lipid oxidation and degradation. Further, the putative autophagic phenotypes of PPARgamma-null cells could be rescued by re-expression of either the PPARgamma1 or -gamma2 isoform. Our paper examines the links between autophagy and PPARgamma-related subcellular and histopathological changes taking place during murine prostatic carcinogenesis.

Extended abstract of a paper presented at Microscopy and Microanalysis 2013 in Indianapolis, Indiana, USA, August 4 – August 8, 2013.

In an effort to reestablish blood flow in blocked coronary arteries, heart attack victims are now routinely treated with thrombolytic (clot busting) drugs. However, thrombolytic therapy fails to restore sufficient flow in approximately 20% of cases. The reasons for these failures are undoubtedly multifactorial and difficult to study in vivo. We have developed an “in vitro” model blood vessel which allows controlled study of factors influencing thrombolysis. In the studies reported here, we investigated the 3-D distribution of HUVEC secreted PAI-1 within the clot using immuno-gold cytochemistry and intermediate voltage electron microscopy (IVEM).Human umbilical vein endothelial cells (HUVEC) were grown on the inside surface of fibronectin-coated glass tubes. The cells are stimulated for 4 hours with 1000 U/ml of TNF-α. Clot formation was initiated by addition of 2.3 μM fibrinogen, 100 nM plasminogen, and then 2 nM thrombin in HEPES buffered Gey's salts + 0.5% human serum albumin. After aging the clot for up to 4 hours (to mimic the time delay between heart attack and treatment), 0.5 nM rt-PA (thrombolytic agent) was perfused through the clot for 10 min. at 5 ml/hr. Formation and dissolution of the clot were monitored by laser light scattering.