Wade Bushman

Etiology, Epidemiology, and Natural History

Historically, benign prostatic hyperplasia (BPH) has been a major focus of urologic practice and surgery. But a simplistic causal relationship among prostatic enlargement, progressive obstruction, lower urinary tract symptoms, retention, and complications of retention has been challenged by recognition of the incomplete overlap of prostatic enlargement with symptoms and obstruction. The result has been a greater focus on symptoms than prostatic enlargement and a shift from surgery to medical treatment. Therefore, the question can be asked whether BPH per se, the glandular enlargement as it contributes to bladder dysfunction, or hyperplastic enlargement as a biomarker for generalized lower urinary tract dysfunction are concerns. This article addresses these issues.

Dose- and Route-Dependent Teratogenicity, Toxicity, and Pharmacokinetic Profiles of the Hedgehog Signaling Antagonist Cyclopamine in the Mouse

The Hedgehog (Hh) signaling pathway is an essential regulator of embryonic development and appears to play important roles in postnatal repair and cancer progression and metastasis. The teratogenic Veratrum alkaloid cyclopamine is a potent Hh antagonist and is used experimentally both in vitro and in vivo to investigate the role of Hh signaling in diverse biological processes. Here, we set out to establish an administration regimen for cyclopamine-induced teratogenicity in the mouse. The dysmorphogenic concentration of cyclopamine was determined in vitro via mouse whole-embryo culture assays to be 2.0 microM. We administered cyclopamine to female C57BL/6J mice at varied doses by oral gavage, ip injection, or osmotic pump infusion and assessed toxicity and pharmacokinetic (PK) models. Bolus administration was limited by toxicity and rapid clearance. In vivo cyclopamine infusion at 160 mg/kg/day yielded a dam serum steady-state concentration of approximately 2 microM with a corresponding amniotic fluid concentration of approximately 1.5 microM. Gross facial defects were induced in 30% of cyclopamine-exposed litters, with affected embryos exhibiting cleft lip and palate. This is the first report describing the PKs and teratogenic potential of cyclopamine in the mouse and demonstrates that transient Hh signaling inhibition induces facial clefting anomalies in the mouse that mimic common human birth defects.

Inactivation of Apc in the Mouse Prostate Causes Prostate Carcinoma

Alterations of the Wnt/beta-catenin signaling pathway are positively associated with the development and progression of human cancer, including carcinoma of the prostate. To determine the role of activated Wnt/beta-catenin signaling in mouse prostate carcinogenesis, we created a mouse prostate tumor model using probasin-Cre-mediated deletion of Apc. Prostate tumors induced by the deletion of Apc have elevated levels of beta-catenin protein and are highly proliferative. Tumor formation is fully penetrant and follows a consistent pattern of progression. Hyperplasia is observed as early as 4.5 weeks of age, and adenocarcinoma is observed by 7 months. Continued tumor growth usually necessitated sacrifice between 12 and 15 months of age. Despite the high proliferation rate, we have not observed metastasis of these tumors to the lymph nodes or other organs. Surgical castration of 6-week-old mice inhibited tumor formation, and castration of mice with more advanced tumors resulted in the partial regression of specific prostate glands. However, significant areas of carcinoma remained 2 months postcastration, suggesting that tumors induced by Apc loss of function are capable of growth under conditions of androgen depletion. We conclude that the prostate-specific deletion of Apc and the increased expression of beta-catenin associated with prostate carcinoma suggests a role for beta-catenin in prostate cancer and offers an appropriate animal model to investigate the interaction of Wnt signaling with other genetic and epigenetic signals in prostate carcinogenesis.

The Sonic Hedgehog pathway stimulates prostate tumor growth by paracrine signaling and recapitulates embryonic gene expression in tumor myofibroblasts.

The Hedgehog (Hh) pathway contributes to prostate cancer growth and progression. The presence of robust Sonic Hedgehog (Shh) expression in both normal prostate and localized cancer challenged us to explain the unique growth-promoting effect in cancer. We show here that paracrine Hh signaling exerts a non-cell autonomous effect on xenograft tumor growth and that Hh pathway activation in myofibroblasts alone is sufficient to stimulate tumor growth. Nine genes regulated by Hh in the mesenchyme of the developing prostate were found to be regulated in the stroma of Hh overexpressing xenograft tumors. Correlation analysis of gene expression in matched specimens of benign and malignant human prostate tissue revealed a partial five-gene fingerprint of Hh-regulated expression in stroma of all cancers and the complete nine-gene fingerprint in the subset of tumors exhibiting a reactive stroma. No expression fingerprint was observed in benign tissues. We conclude that changes in the prostate stroma due to association with cancer result in an altered transcriptional response to Hh that mimics the growth-promoting actions of the fetal mesenchyme. Patients with an abundance of myofibroblasts in biopsy tissue may comprise a subgroup that will exhibit a particularly good response to anti-Hh therapy.

HOXA-13 GENE MUTATION RESULTS IN ABNORMAL SEMINAL VESICLE AND PROSTATE DEVELOPMENT

The role of Hoxa-13 in postnatal morphogenesis of the male accessory sex organs was assessed by correlating the Hoxa-13 expression domain with phenotypic abnormalities in heterozygous Hypodactyly mutants. Hypodactyly is a naturally occurring semi-dominant mutation that results from a 50-base pair deletion in exon one of the Hoxa-13 allele. We demonstrate that Hoxa-13 is broadly expressed in the developing lower genitourinary tract and that the Hypodactyly mutation results in a specific phenotype characterized by decreased size and branching of the dorsolateral and ventral prostate and abnormal seminal vesicle morphology. This phenotype partially overlaps the genitourinary phenotype observed in Hoxd-13 deficient mice and comparison showed similar domains of Hoxa-13 and Hoxd-13 expression in the lower genitourinary tract. The similarity in expression and overlap in phenotype resulting from mutation is consistent with additive function and partial functional redundancy of Hoxa-13 and Hoxd-13 in male accessory sex organ development.

Blockade of NGF and trk receptors inhibits increased peripheral mechanical sensitivity accompanying cystitis in rats

Visceral inflammation, including that arising from bladder inflammation, reduces the threshold to sensation of innocuous or noxious stimuli applied to peripheral structures (referred hyperalgesia). Cystitis may induce transient or persistent plastic changes mediated by neurotrophins, particularly nerve growth factor (NGF), which contribute to increased nociceptive input. In this study, acute or subacute cystitis was induced in female rats by one or three (at 72-h intervals) 400-microl intravesical instillations of 1 mM acrolein. Sensitivity of the hindpaws to mechanical and thermal stimuli was determined before and 4, 24, 48, 72, and 96 h after treatment. Other groups of rats were treated with intravesical or intrathecal k252a [a nonspecific antagonist of tyrosine kinase (trk) receptors, including trkA, the high-affinity receptor for NGF] before the first or third acrolein instillation. Some rats were intraperitoneally injected with specific NGF-neutralizing antiserum or normal serum before acrolein instillation. Acute and subacute cystitis induced mechanical, but not thermal, referred hyperalgesia that was attenuated by intravesical pretreatment with k252a. Systemic treatment with NGF-neutralizing antiserum before instillation of acrolein suppressed subsequent mechanical referred hyperalgesia. Expression of NGF was increased within the bladder by acute or subacute cystitis and in L6/S1 dorsal root ganglia by subacute cystitis. These results suggest that the bladder-derived NGF acting via trk receptors at least partially mediates peripheral sensitization to mechanical stimuli associated with acute and subacute acrolein-induced cystitis.

Suspended microfluidics

Although the field of microfluidics has made significant progress in bringing new tools to address biological questions, the accessibility and adoption of microfluidics within the life sciences are still limited. Open microfluidic systems have the potential to lower the barriers to adoption, but the absence of robust design rules has hindered their use. Here, we present an open microfluidic platform, suspended microfluidics, that uses surface tension to fill and maintain a fluid in microscale structures devoid of a ceiling and floor. We developed a simple and ubiquitous model predicting fluid flow in suspended microfluidic systems and show that it encompasses many known capillary phenomena. Suspended microfluidics was used to create arrays of collagen membranes, mico Dots (μDots), in a horizontal plane separating two fluidic chambers, demonstrating a transwell platform able to discern collective or individual cellular invasion. Further, we demonstrated that μDots can also be used as a simple multiplexed 3D cellular growth platform. Using the μDot array, we probed the combined effects of soluble factors and matrix components, finding that laminin mitigates the growth suppression properties of the matrix metalloproteinase inhibitor GM6001. Based on the same fluidic principles, we created a suspended microfluidic metabolite extraction platform using a multilayer biphasic system that leverages the accessibility of open microchannels to retrieve steroids and other metabolites readily from cell culture. Suspended microfluidics brings the high degree of fluidic control and unique functionality of closed microfluidics into the highly accessible and robust platform of open microfluidics.

Models of Inflammation of the Lower Urinary Tract

Inflammation of the lower urinary tract occurs frequently in people. The causes remain obscure, with the exception of urinary tract infection. Animal models have proven useful for investigating and assessing mechanisms underlying symptoms associated with lower urinary tract inflammation and options for suppressing these symptoms. This review will discuss various animal models of lower urinary tract inflammation, including feline spontaneous (interstitial) cystitis, neurogenic cystitis, autoimmune cystitis, cystitis induced by intravesical instillation of chemicals or bacterial products (particularly lipopolysaccharide or LPS), and prostatic inflammation initiated by transurethral instillation of bacteria. Animal models will continue to be of significant value in identifying mechanisms resulting in bladder inflammation, but the relevance of some of these models to the causes underlying clinical disease is unclear. This is primarily because of the lack of understanding of causes of these disorders in people. Comparative and translational studies are required if the full potential of findings obtained with animal models to improve prevention and treatment of lower urinary tract inflammation in people is to be realized. Neurourol. Urodynam. 30:673–682, 2011.

Acute bacterial inflammation of the mouse prostate

Prostatic inflammation is gaining increasing attention as a potential etiologic factor in prostate cancer, benign prostatic hyperplasia, lower urinary tract symptoms, and CPPS. This study was performed to address the need for a well characterized model of acute prostatic inflammation that may be used to study the effect of acute inflammation on epithelial and stromal cell proliferation, voiding behavior, and neurovascular physiology.

Sonic Hedgehog-responsive Genes in the Fetal Prostate

The Hedgehog (Hh) signaling pathway plays an important role in prostate development and appears to play an equally important role in promoting growth of advanced prostate cancer. During prostate development, epithelial cells in the urogenital sinus (UGS) express Sonic Hedgehog (Shh) and secrete Shh peptide. The secreted Hh peptide acts on adjacent mesenchymal cells to activate the Hh signal transduction pathway and elicit paracrine effects on epithelial proliferation and differentiation. To identify mesenchymal targets of Shh signaling, we performed microarray analysis on a Shh-responsive, immortalized urogential sinus mesenchymal cell line. We found 68 genes that were up-regulated by Shh and 21 genes that were down-regulated. Eighteen of those were selected for further study with Ptc1 and Gli1 serving as reference controls. We found 10 of 18 were also Hh-regulated in primary UGS mesenchymal cells and 13 of 18 in the cultured UGS. Seven of 18 exhibited Shh-regulated expression in both assays (Igfbp-6, Igfbp-3, Fbn2, Ntrk3, Agpt4, Dmp1, and Mmp13). Three of the 18 genes contained putative Gli binding motifs that bound Gli1 peptide in electrophoretic mobility shift assays. With the exception of Tiam1, target gene expression generally showed no differences in the concentration dependence of ligand-induced expression, but we observed strikingly different responses to direct pathway activation by transfection with activated Smo, Gli1, and Gli2.