Brian J. Philips

Differential Expression of Functional Cannabinoid Receptors in Human Bladder Detrusor and Urothelium

PURPOSE Although cannabinoid receptor expression has been demonstrated in human brain and other peripheral neuronal tissues, definitive expression of these receptors in the human bladder has not been reported. Consequently we investigated the expression of functional cannabinoid 1 and 2 receptors in human bladder detrusor and urothelium. MATERIALS AND METHODS Human bladders were micro-dissected for detrusor (6) and urothelium (8), and analyzed for cannabinoid 1 and 2 mRNA expression using real-time quantitative polymerase chain reaction, and for protein expression using immunohistochemistry and Western blot. Functional response of these receptors was tested by studying the effect of selective cannabinoid 1 and 2 agonists on nerve evoked smooth muscle contraction. RESULTS Quantitative polymerase chain reaction analysis revealed differential expression of cannabinoid 1 and 2 receptors in detrusor and urothelium. The expression of cannabinoid 1 and 2 receptor mRNA in urothelium was approximately 2-fold higher than in detrusor, although this was not significant (p >0.05). Cannabinoid 1 receptor mRNA expression was significantly higher than cannabinoid 2 receptor expression in the 2 tissue subtypes (p </=0.05). Expression at mRNA level was confirmed at the protein level by immunoreactivity and Western blot analysis. Activation of cannabinoid 1 and 2 receptors in human bladder attenuated the electrically evoked contraction of detrusor strips. CONCLUSIONS Together these findings suggest a physiological role of cannabinoid 1 and 2 receptors in the human bladder. Moreover, these results confirm the presence of functional cannabinoid 1 and 2 receptors in the human bladder, which can serve as a target for drugs acting on symptoms of interstitial cystitis/painful bladder syndrome.

Antioxidant effects of green tea and its polyphenols on bladder cells

Genitourinary tract inflammation/ailments affect the quality of life and health of a large segment of society. In recent years, studies have demonstrated strong antioxidant effects of green tea and its associated polyphenols in inflammatory states. This in vitro study examined the antioxidant capabilities (and putative mechanisms of action) of green tea extract (GTE), polyphenon-60 (PP-60, 60% pure polyphenols), (-)-epicatechin-3-gallate (ECG) and (-)-epigallocatechin-3-gallate (EGCG) in normal/malignant human bladder cells following catechin treatment+/-1 mM H2O2 (oxidative agent). Cell viability, apoptosis and reactive oxygen species (ROS) formation were evaluated. Our results showed that H2O2 exposure significantly reduced normal (UROtsa) and high-grade (TCCSUP, T24) bladder cancer (BlCa) cell viability compared with control-treated cells (p<0.001). No affect on low-grade RT4 and SW780 BlCa cell viability was observed with exposure to H2O2. Compared to H2O2-treated UROtsa, treatment with PP-60, ECG and EGCG in the presence of H2O2 significantly improved UROtsa viability (p<0.01), with strongest effects evoked by ECG. Additionally, though not as effective as in UROtsa cells, viability of both high-grade TCCSUP and T24 BlCa cells, in comparison to H2O2-treated cells, was significantly improved (p<0.01) by treatment with PP-60, ECG, and EGCG in the presence of H2O2. Overall, our findings demonstrate that urothelium cell death via H2O2-induced oxidative stress is mediated, in part, through superoxide (O2-.;), and potentially, direct H2O2 mechanisms, suggesting that green tea polyphenols can protect against oxidative stress/damage and bladder cell death.

Multiplex Analysis of Urinary Cytokine Levels in Rat Model of Cyclophosphamide-induced Cystitis

OBJECTIVES The urinary proteome is a potential easily accessible source of biomarkers for inflammatory bladder diseases, including interstitial cystitis. In the present study, we subjected rat urine to multiplex cytokine analysis in an attempt to identify an inflammatory signature of the temporal course of cyclophosphamide (CYP)-induced cystitis. METHODS Rat urine was collected for 12 hours after CYP injection (150 mg/kg) for multiplex analysis of 14 cytokines by a multiple antigen bead assay (Luminex 100 IS). Urine from each void was collected, and the voiding frequency was determined. The bladder tissue was analyzed for cytokines levels and histologic evidence of inflammation. RESULTS Significant changes were noted in the urine levels of all cytokines with respect to baseline at 2, 4, 6, and 10 hours after CYP injection. Elevation was noted at all times for most cytokines, except for monocyte chemotactic protein-1, which had a 5-fold decrease at 2 hours. The urine and tissue levels of interleukin (IL)-1beta, IL-4, and growth-related oncogene/keratinocyte-derived chemokine correlated significantly, with a positive Spearman correlation also noted for granulocyte-macrophage colony-stimulating factor, monocyte chemotactic protein-1-1, IL-18, and interferon-gamma. The tissue levels for most cytokines, except for IL-2, and urinary frequency were significantly elevated in the CYP-treated rats compared with the control vehicle-treated rats. The hints of severe inflammation in the bladder indicated by the urinary cytokines were confirmed by bladder histologic examination and the tissue cytokine levels at necropsy. CONCLUSIONS The progression of CYP-induced cystitis was clearly reflected in the urine matrix by the temporal and quantitative changes in the cytokine levels. Additional delineation of urine and bladder tissue cytokine expression might yield biomarkers for cystitis.

Prevalence of endogenous CD34+ adipose stem cells predicts human fat graft retention in a xenograft model.

Background: Fat grafting is a promising technique for soft-tissue augmentation, although graft retention is highly unpredictable and factors that affect graft survival have not been well defined. Because of their capacity for differentiation and growth factor release, adipose-derived stem cells may have a key role in graft healing. The authors’ objective was to determine whether biological properties of adipose-derived stem cells present within human fat would correlate with in vivo outcomes of graft volume retention. Methods: Lipoaspirate from eight human subjects was processed using a standardized centrifugation technique and then injected subcutaneously into the flanks of 6-week-old athymic nude mice. Graft masses and volumes were measured, and histologic evaluation, including CD31+ staining for vessels, was performed 8 weeks after transplantation. Stromal vascular fraction isolated at the time of harvest from each subject was analyzed for surface markers by multiparameter flow cytometry, and also assessed for proliferation, differentiation capacity, and normoxic/hypoxic vascular endothelial growth factor secretion. Results: Wide variation in percentage of CD34+ progenitors within the stromal vascular fraction was noted among subjects and averaged 21.3 ± 15 percent (mean ± SD). Proliferation rates and adipogenic potential among stromal vascular fraction cells demonstrated moderate interpatient variability. In mouse xenograft studies, retention volumes ranged from approximately 36 to 68 percent after 8 weeks, with an overall average of 52 ± 11 percent. A strong correlation (r = 0.78, slope = 0.76, p < 0.05) existed between stromal vascular fraction percentage of CD34+ progenitors and high graft retention. Conclusion: Inherent biological differences in adipose tissue exist between patients. In particular, concentration of CD34+ progenitor cells within the stromal vascular fraction may be one of the factors used to predict human fat graft retention.

Adipose stem cell-based soft tissue regeneration

Introduction: Since their isolation and characterization nearly a decade ago, adipose-derived stem cells (ASCs) have become one of the most popular adult stem cell populations for research in soft tissue engineering and regenerative medicine applications. Compared with other stem cell sources, ASCs offer several advantages including an abundant autologous source, minor invasive harvesting (liposuction), significant proliferative capacity in culture and multi-lineage potential. Numerous preclinical studies have been pursued, with early clinical data appearing in the literature. Areas covered: Autologous fat grafting has gained tremendous momentum in clinical practice over the past several years due to its potential applications in trauma and reconstructive surgery. This review focuses on the published clinical and pre-clinical (i.e., animal) data to date using ASCs for soft tissue reconstruction, with particular attention to experimental models and methodologies. Future directions for rendering soft tissue reconstructive therapies more effective are discussed. Expert opinion: Although standardization of ASC harvesting and processing techniques, as well as long-term results of existing clinical studies, remains to be addressed, the known biological properties of ASCs suggest a potential role in enhancing fat graft retention and facilitating minimally invasive reconstructive treatments. While clinical applications are being reported, well controlled clinical studies are needed to demonstrate safety and efficacy.

Estrogen Sulfotransferase Inhibits Adipocyte Differentiation

The estrogen sulfotransferase (EST) is a phase II drug-metabolizing enzyme known to catalyze the sulfoconjugation of estrogens. EST is highly expressed in the white adipose tissue of male mice, but the role of EST in the development and function of adipocytes remains largely unknown. In this report, we showed that EST played an important role in adipocyte differentiation. EST was highly expressed in 3T3-L1 preadipocytes and primary mouse preadipocytes. The expression of EST was dramatically reduced in differentiated 3T3-L1 cells and mature primary adipocytes. Overexpression of EST in 3T3-L1 cells prevented adipocyte differentiation. In contrast, preadipocytes isolated from EST knockout (EST-/-) mice exhibited enhanced differentiation. The inhibitory effect of EST on adipogenesis likely resulted from the sustained activation of ERK1/2 MAPK and inhibition of insulin signaling, leading to a failure of switch from clonal expansion to differentiation. The enzymatic activity of EST was required for the inhibitory effect of EST on adipogenesis, because an enzyme-dead EST mutant failed to inhibit adipocyte differentiation. In vivo, overexpression of EST in the adipose tissue of female transgenic mice resulted in smaller adipocyte size. Taken together, our results suggest that EST functions as a negative regulator of adipogenesis.

Spatially controlled delivery of neurotrophic factors in silk fibroin-based nerve conduits for peripheral nerve repair.

Restoration with sufficient functional recovery after long-gap peripheral nerve damage remains a clinical challenge. Silk has shown clinical promise for numerous tissue engineering applications due to its biocompatibility, impressive mechanical properties, and Food and Drug Administration approval. The aim of this study was to evaluate the efficacy of silk fibroin–based nerve guides containing glial cell line-derived neurotrophic factor (GDNF) in a long-gap sized (15 mm) rat sciatic nerve defect model. Four groups of nerve conduits were prepared: (1) silk conduits with empty silk microspheres, (2) silk conduits with GDNF-loaded silk microspheres uniformly distributed in the conduit wall, (3) silk conduits with GDNF-loaded silk microspheres in a controlled manner with the highest GDNF concentration at the distal end, and (4) isograft. After 6 weeks, the nerve grafts were explanted, harvested, and fixed for histologic analysis. Nerve tissue stained with the S-100, and neuroendocrine marker PGP 9.5 antibodies demonstrated a significantly increased density of nerve tissue in the GDNF-treated groups compared with the empty microsphere (control) group (P < 0.05). GDNF-treated animals with a higher concentration of GDNF in the distal portion possessed a significantly higher density of PGP 9.5 protein middle conduit part than comparison to GDNF uniform-treated animals (P < 0.05). Silk-based nerve conduits possess optimal mechanical and degradative properties, rendering them potentially useful in peripheral nerve repair. This study demonstrates that novel, porous silk fibroin–based nerve conduits, infused with GDNF in a controlled manner, represent a potentially viable conduit for Schwann cell migration and proliferation in the regeneration of peripheral nerves.

Estrogen Sulfotransferase/SULT1E1 Promotes Human Adipogenesis

ABSTRACT Estrogen sulfotransferase (EST/SULT1E1) is known to catalyze the sulfoconjugation and deactivation of estrogens. The goal of this study is to determine whether and how EST plays a role in human adipogenesis. By using human primary adipose-derived stem cells (ASCs) and whole-fat tissues from the abdominal subcutaneous fat of obese and nonobese subjects, we showed that the expression of EST was low in preadipocytes but increased upon differentiation. Overexpression and knockdown of EST in ASCs promoted and inhibited differentiation, respectively. The proadipogenic activity of EST in humans was opposite to the antiadipogenic effect of the same enzyme in rodents. Mechanistically, EST promoted adipogenesis by deactivating estrogens. The proadipogenic effect of EST can be recapitulated by using an estrogen receptor (ER) antagonist or ERα knockdown. In contrast, activation of ER in ASCs inhibited adipogenesis by decreasing the recruitment of the adipogenic peroxisome proliferator-activated receptor γ (PPARγ) onto its target gene promoters, whereas ER antagonism increased the recruitment of PPARγ to its target gene promoters. Linear regression analysis revealed a positive correlation between the expression of EST and body mass index (BMI), as well as a negative correlation between ERα expression and BMI. We conclude that EST is a proadipogenic factor which may serve as a druggable target to inhibit the turnover and accumulation of adipocytes in obese patients.

Healing of Grafted Adipose Tissue: Current Clinical Applications of Adipose-Derived Stem Cells for Breast and Face Reconstruction

Since their isolation and characterization nearly a decade ago, adipose‐derived stem cells (ASCs) have become one of the most popular adult stem cell populations for soft tissue engineering and regenerative medicine applications. Compared with other stem cell sources, ASCs offer several advantages including abundant autologous source, minor invasive harvesting (liposuction), significant proliferative capacity in culture, and multilineage potential. In this mini review, we focus on some of the more salient published clinical and preclinical data to date regarding ASC treatment for breast and facial soft tissue reconstruction.

Polyunsaturated fatty acid metabolizing 15-Lipoxygenase-1 (15-LO-1) expression in normal and tumorigenic human bladder tissues.

Diets high in fat seem to correspond with an increased risk of certain forms of cancer, including bladder BlCa. This preliminary study examined the expression and enzyme activity profile of the polyunsaturated fatty acid metabolizing enzyme 15-Lipoxygenase-1 (15-LO-1) in human tissues from normal bladder and bladder tumors (stages CIS-T3/T4). Human tissue samples from normal (donor) bladder and bladder tumors (stages CIS-T3/T4; non–Bacillus Calmette-Guerin-treated) were grossly microdissected and analyzed for 15-LO-1 protein expression [immunohistochemistry (IHC)/Western blot], mRNA expression (quantitative real-time polymerase chain reaction) and enzyme activity profiles. Our results demonstrated that 15-LO-1 expression (protein/mRNA) and enzyme activity varied with BlCa progression. Specifically, IHC analyses of 15-LO-1 protein levels revealed decreased expression with increased bladder tumor stage. In particular, a statistically significant decrease in 15-LO-1 expression in stage T3/T4 bladder tumors compared with normal tissues (P<0.001) was observed. In agreement with IHC results, Western blot, quantitative real-time polymerase chain reaction, and enzymatic activity analyses demonstrated increased 15-LO-1 protein, mRNA, and enzyme activity, respectively, in normal human bladder tissues in comparison with stage T3/T4 human bladder tumors. Our finding of variable 15-LO-1 expression and enzyme activity in bladder tissues suggests a role for 15-LO-1 in bladder carcinogenesis.