In Vivo Longitudinal Monitoring of Cardiac Remodeling in Murine Ischemia Models With Adaptive Bayesian Regularized Cardiac Strain Imaging: Validation Against Histology.

Adaptive Bayesian regularized cardiac strain imaging (ABR-CSI) uses raw radiofrequency signals to estimate myocardial wall contractility as a surrogate measure of relative tissue elasticity incorporating regularization in the Bayesian sense. We determined the feasibility of using ABR-CSI -derived strain for in vivo longitudinal monitoring of cardiac remodeling in a murine ischemic injury model (myocardial infarction [MI] and ischemia-reperfusion [IR]) and validated the findings against ground truth histology. We randomly stratified 30 BALB/CJ mice (17 females, 13 males, median age = 10 wk) into three surgical groups (MI = 10, IR = 12, sham = 8) and imaged pre-surgery (baseline) and 1, 2, 7 and 14 d post-surgery using a pre-clinical high-frequency ultrasound system (VisualSonics Vevo 2100). We then used ABR-CSI to estimate end-systolic and peak radial (er) and longitudinal (el) strain estimates. ABR-CSI was found to have the ability to serially monitor non-uniform cardiac remodeling associated with murine MI and IR non-invasively through temporal variation of strain estimates post-surgery. Furthermore, radial end-systole (ES) strain images and segmental strain curves exhibited improved discrimination among infarct, border and remote regions around the myocardium compared with longitudinal strain results. For example, the MI group had significantly lower (Friedman's with Bonferroni-Dunn test, p = 0.002) ES er values in the anterior middle (infarcted) region at day 14 (n = 9, 9.23 ± 7.39%) compared with the BL group (n = 9, 44.32 ± 5.49). In contrast, anterior basal (remote region) mean ES er values did not differ significantly (non-significant Friedman's test, χ2 = 8.93, p = 0.06) at day 14 (n = 6, 33.05 ± 6.99%) compared with baseline (n = 6, 34.02 ± 6.75%). Histology slides stained with Masson's trichrome (MT) together with a machine learning model (random forest classifier) were used to derive the ground truth cardiac fibrosis parameter termed histology percentage of myocardial fibrosis (PMF). Both radial and longitudinal strain were found to have strong statistically significant correlations with the PMF parameter. However, radial strain had a higher Spearman's correlation value (εresρ = -0.67, n = 172, p < 0.001) compared with longitudinal strain (εlesρ = -0.60, n = 172, p < 0.001). Overall, the results of this study indicate that ABR-CSI can reliably perform non-invasive detection of infarcted and remote myocardium in small animal studies.

Murine cardiac fibrosis localization using adaptive Bayesian cardiac strain imaging in vivo.

An adaptive Bayesian regularized cardiac strain imaging (ABR-CSI) algorithm for in vivo murine myocardial function assessment is presented. We report on 31 BALB/CJ mice (n = 17 females, n = 14 males), randomly stratified into three surgical groups: myocardial infarction (MI, n = 10), ischemia-reperfusion (IR, n = 13) and control (sham, n = 8) imaged pre-surgery (baseline- BL), and 1, 2, 7 and 14 days post-surgery using a high frequency ultrasound imaging system (Vevo 2100). End-systole (ES) radial and longitudinal strain images were used to generate cardiac fibrosis maps using binary thresholding. Percentage fibrotic myocardium (PFM) computed from regional fibrosis maps demonstrated statistically significant differences post-surgery in scar regions. For example, the MI group had significantly higher PFMRadial (%) values in the anterior mid region (p = 0.006) at Day 14 (n = 8, 42.30 ± 14.57) compared to BL (n = 12, 1.32 ± 0.85). A random forest classifier automatically detected fibrotic regions from ground truth Masson's trichrome stained histopathology whole slide images. Both PFMRadial (r = 0.70) and PFMLongitudinal (r = 0.60) results demonstrated strong, positive correlation with PFMHistopathology (p < 0.001).

Bayesian Regularized Strain Imaging for Assessment of Murine Cardiac Function In vivo.

A cardiac strain imaging framework with adaptive Bayesian regularization (ABR) is proposed for in vivo assessment of murine cardiac function. The framework uses ultrasound (US) radio-frequency data collected with a high frequency (fc = 30MHz) imaging system and a multi-level block matching algorithm with ABR to derive inter-frame cardiac displacements. Lagrangian cardiac strain (radial, er and longitudinal, el) tensors were derived by segmenting the myocardial wall starting at the ECG R-wave and accumulating interframe deformations over a cardiac cycle. In vivo feasibility was investigated through a longitudinal study with two mice (one ischemia-perfusion (IR) injury and one sham) imaged at five sessions (pre-surgery (BL) and 1,2,7 and 14 days post-surgery). End-systole (ES) strain images and segmental strain curves were derived for quantitative evaluation. Both mice showed periodic variation of er and el strain at BL with segmental synchroneity. Infarcted regions of IR mouse at Day 14 were associated with reduced or sign reversed ES er and el values while the sham mouse had similar or higher strain than at BL. Infarcted regions identified in vivo were associated with increased collagen content confirmed with Masson's Trichrome stained ex vivo heart sections.Clinical Relevance-Higher quality cardiac strain images derived with RF data and Bayesian regularization can potentially improve the sensitivity and accuracy of non-invasive assessment of cardiovascular disease models.

Finasteride treatment alters tissue specific androgen receptor expression in prostate tissues.

BACKGROUND: Normal and pathologic growth of the prostate is dependent on the synthesis of dihydrotestosterone (DHT) from testosterone by 5α-reductase. Finasteride is a selective inhibitor of 5α-reductase 2, one isozyme of 5α-reductase found in abundance in the human prostate. The objective of this study was to investigate the effects of finasteride on androgen receptor expression and tissue morphology in human benign prostatic hyperplasia specimens. METHODS: Patients undergoing transurethral resection of the prostate and either treated or not treated with finasteride between 2004 and 2010 at the University of Wisconsin-Hospital were retrospectively identified using an institutional database. Prostate specimens from each patient were triple-stained for androgen receptor, prostate-specific antigen, and basal marker cytokeratin 5. Morphometric analysis was performed using the multispectral imaging, and results were compared between groups of finasteride treated and non-treated patients. RESULTS: Epithelial androgen receptor but not stromal androgen receptor expression was significantly lower in patients treated with finasteride than in non-treated patients. Androgen receptor-regulated prostate-specific antigen was not significantly decreased in finasteride-treated patients. Significant luminal epithelial atrophy and basal cell hyperplasia were prevalent in finasteride treated patients. Epithelial androgen receptor expression was highly correlated to the level of luminal epithelial atrophy. CONCLUSIONS: In this study, finasteride decreased the expression of epithelial androgen receptor in a tissue specific manner. The correlation between epithelial androgen receptor and the extent of luminal epithelial atrophy suggests that epithelial androgen receptor may be directly regulating the atrophic effects observed with finasteride treatment.

Angiogenesis induced by signal transducer and activator of transcription 5A (STAT5A) is dependent on autocrine activity of proliferin.

Multiple secreted factors induce the formation of new blood vessels (angiogenesis). The signal transduction events that orchestrate the numerous cellular activities required for angiogenesis remain incompletely understood. We have shown previously that STAT5 plays a pivotal role in angiogenesis induced by FGF2 and FGF8b. To delineate the signaling pathway downstream of STAT5, we expressed constitutively active (CA) or dominant-negative (DN) mutant STAT5A in mouse brain endothelial cells (EC). We found that the conditioned medium from CA-STAT5A but not from dominant-negative STAT5A overexpressing EC is sufficient to induce EC invasion and tube formation, indicating that STAT5A regulates the secretion of autocrine proangiogenic factors. Conversely, CA-STAT5A-induced conditioned medium had no effect on EC proliferation. Using a comparative genome-wide transcription array screen, we identified the prolactin family member proliferin (PLF1 and PLF4) as a candidate autocrine factor. The CA-STAT5A-dependent transcription and secretion of PLF by EC was confirmed by quantitative RT-PCR and Western blotting, respectively. CA-STAT5A binds to the PLF1 promoter region, suggesting a direct transcriptional regulation. Knockdown of PLF expression by shRNA or by blocking of PLF activity with neutralizing antibodies removed the CA-STAT5A-dependent proangiogenic activity from the conditioned medium of EC. Similarly, the ability of concentrated conditioned medium from CA-STAT5A transfected EC to induce angiogenesis in Matrigel plugs in vivo was abolished when PLF was depleted from the medium. These observations demonstrate a FGF/STAT5/PLF signaling cascade in EC and implicate PLF as autocrine regulator of EC invasion and tube formation.

Protein expression of matriptase and its cognate inhibitor HAI-1 in human prostate cancer: a tissue microarray and automated quantitative analysis.

Recent studies have suggested that matriptase, a transmembrane serine protease and its cognate inhibitor hepatocyte growth factor activator inhibitor-1 (HAI-1) are important in the progression of many cancers. Limited quantitative data are available on these proteins in prostate cancer. To validate the roles of matriptase and HAI-1 in prostate cancer and its progression, a prostate cancer tissue microarray was constructed. The tissue microarray includes 41 localized prostate cancers (Pca_local), 18 aggressive prostate cancers, 18 metastatic prostate cancers, 24 benign prostate hyperplasias, 18 high-grade intraepithelial neoplasias (HGPIN), and 41 benign prostate tissues. The cellular expression levels of matriptase and HAI-1 were quantified using automated quantitative analysis. We found that matriptase expression levels were significantly higher in Pca_local (P<0.0001) and HGPIN (P<0.05) compared with benign prostate tissue. Matriptase levels were significantly decreased in metastatic cancer when compared with all other tissue types (P<0.05). Compared with benign prostate tissue, HAI-1 expression levels were significantly higher in all proliferative prostate diseases (benign prostate hyperplasia, HGPIN, localized and aggressive cancers, and metastases) (P<0.001); yet, no significant differences were found in HAI-1 expression levels among the diseased tissue types. These results suggest that an increase of matriptase may be useful as a marker for detection of Pca_local, whereas a decrease of matriptase expression may signal prostate cancer progression. HAI-1 seems to be a marker of prostate epithelial cell proliferation.

Oncocytoma can be differentiated from its renal cell carcinoma mimics by a panel of markers: an automated tissue microarray study.

BACKGROUND: Differentiating oncocytoma from its renal cell carcinoma (RCC) mimics, particularly chromophobe RCC, can be difficult, especially when limited tissue is available for evaluation. This study presents a panel of markers that are readily available, easy to use, and useful for differential diagnoses of renal tumors. DESIGN: A renal cell neoplasm tissue microarray was constructed including oncocytoma (n=30), chromophobe RCC (n=18), conventional RCC (n=64), papillary RCC (n=50), and benign renal tissues (n=31). CK7, CD10, epithelial membrane antigen, renal cell carcinoma marker (RCCma), vimentin, and endogenous avidin-binding activity (EABA) were studied. An Automated Cellular Imaging System, was used to quantify the staining intensity. RESULT: EABA was positive in 97% of oncocytoma, 26% of conventional RCC and 35% of papillary RCC with granular/eosinophilic (G/E) features and 6% of chromophobe RCC. EABA was negative in RCC without G/E features. Vimentin and RCCma were positive in most RCC with G/E features (conventional, 78% and 71%; and papillary, 85% and 76%, respectively), and negative in oncocytoma. Vimentin was also negative in chromophobe RCC. CK7 was positive in up to 81% of papillary RCC and 63% of chromophobe RCC, and essentially negative in conventional RCC and oncocytoma. CONCLUSIONS: EABA is an excellent marker for oncocytoma, which can be useful in differentiating oncocytoma from chromophobe RCC. A panel of EABA, vimentin, and RCCma markers can be useful in discerning oncocytoma from RCC with G/E features. Vimentin can be useful in discriminating chromophobe RCC from papillary or conventional RCCs.

High endogenous avidin binding activity: an inexpensive and readily available marker for the differential diagnosis of kidney neoplasms.

It has been documented that some tissues, such as salivary gland, liver, cardiac and skeletal muscles and kidney, have high level endogenous biotin or endogenous avidin binding activity (EABA). Limited data is available on EABA in renal cell neoplasms. A tissue microarray (TMA) was constructed that included oncocytoma (n=30), chromophobe renal cell carcinoma (RCC) (n=18), clear cell RCC (n=45), clear cell RCC with granular/eosinophilic (G/E) features (n=19), papillary RCC (n=21), papillary RCC with G/E features (n=29) and benign renal tissues (n=31). The TMA slides were stained with or without biotin blocker and analyzed using the automated cellular imaging system (ACIS(R)). Without biotin blocker, a high positive rate of EABA was found in oncocytoma (56/60, 93%) and normal renal tubules (46/60, 77%). A moderate positive rate of EABA was found in clear cell and papillary RCCs with G/E features (13/39, 33% and 19/55, 35%, respectively). Chromophobe RCC and RCC without G/E features had essentially no EABA. With biotin blocker, benign renal tissue and clear cell RCC were negative for EABA; but a significant number of renal oncocytoma (29/60, 48%) and a few papillary RCC with G/E features (5/52, 10%) remained positive for EABA. In conclusion, high EABA may be used to differentiate oncocytoma from chromophobe RCC, and the staining results must be interpreted with caution when avidin-biotin detection system is used in diagnosing renal neoplasms.

Effects of a monoclonal anti-alphavbeta3 integrin antibody on blood vessels - a pharmacodynamic study.

PURPOSE: The integrin alphavbeta3 is an adhesion molecule expressed by proliferating endothelial cells and antibodies blocking this integrin inhibit angiogenesis in preclinical models. MEDI-522 is a second generation humanized anti-alphavbeta3 antibody designed for antiangiogenic therapy. The purpose of this study was to examine potential effects of this agent on blood vessels. EXPERIMENTAL DESIGN: In a phase I dose escalation study, MEDI-522 was administered by weekly infusions to 25 adult patients with advanced solid organ malignancies. As a surrogate angiogenesis assay, a wound was created by punch biopsy of the arm skin. This wound site was re-biopsied after a 7-day interval. Dual-label immunofluorescence experiments followed by computer-assisted image analysis were conducted to analyze the vasculature. RESULTS: Sequential pretreatment and 4-week treatment skin biopsy pairs were available on 4 patients, who had received 6 or 10 mg/kg of MEDI-522. MEDI-522 was detected in the dermal blood vessels as well as the dermal interstitium both in intact and wounded skin sites following treatment. No statistically significant difference was found between pretreatment and treatment samples of skin for vascular area, endothelial cell proliferation and apoptosis, or beta3 integrin levels. Phosphorylated focal adhesion kinase (pFAK) was significantly diminished in skin wound vessels during MEDI-522 treatment compared to the pretreatment samples. CONCLUSIONS: MEDI-522 was detectable both in quiescent and in angiogenically active skin blood vessels as well as in the dermal interstitial space. The levels of pFAK were reduced during MEDI-522 treatment, suggesting a modulating effect on this signaling molecule.