Rogely W. Boyce

Activation of Peroxisome Proliferator–Activated Receptor-α Protects the Heart From Ischemia/Reperfusion Injury

Background—Peroxisome proliferator–activated receptor-&agr; (PPAR-&agr;) is expressed in the heart and regulates genes involved in myocardial fatty acid oxidation (FAO). The role of PPAR-&agr; in acute ischemia/reperfusion myocardial injury remains unclear. Methods and Results—The coronary arteries of male mice were ligated for 30 minutes. After reperfusion for 24 hours, ischemic and infarct sizes were determined. A highly selective and potent PPAR-&agr; agonist, GW7647, was administered by mouth for 2 days, and the third dose was given 1 hour before ischemia. GW7647 at 1 and 3 mg · kg−1 · d−1 reduced infarct size by 28% and 35%, respectively (P <0.01), and myocardial contractile dysfunction was also improved. Cardioprotection by GW7647 was completely abolished in PPAR-&agr;–null mice. Ischemia/reperfusion downregulated mRNA expression of cardiac PPAR-&agr; and FAO enzyme genes, decreased myocardial FAO enzyme activity and in vivo cardiac fat oxidation, and increased serum levels of free fatty acids. All of these changes were reversed by GW7647. Moreover, GW7647 attenuated ischemia/reperfusion-induced release of multiple proinflammatory cytokines and inhibited neutrophil accumulation and myocardial expression of matrix metalloproteinases-9 and -2. Furthermore, GW7647 inhibited nuclear factor-&kgr;B activation in the heart, accompanied by enhanced levels of inhibitor-&kgr;B&agr;. Conclusions—Activation of PPAR-&agr; protected the heart from reperfusion injury. This cardioprotection might be mediated through metabolic and antiinflammatory mechanisms. This novel effect of the PPAR-&agr; agonist could provide an added benefit to patients treated with PPAR-&agr; activators for dyslipidemia.

Preliminary comparison of quantity, quality, and microarray performance of RNA extracted from formalin-fixed, paraffin-embedded, and unfixed frozen tissue samples.

Microarrays have been used to simultaneously monitor the expression of thousands of genes from biological samples, an approach that can potentially uncover previously unrecognized functions of genes. Microarray analyses can rarely be conducted retrospectively because of the requirement for RNA to be obtained from fresh or unfixed frozen tissues. Archived pathology specimens would need to be used for retrospective analyses, and these are typically preserved as formalin-fixed, paraffin-embedded (FFPE) tissue. Formalin-fixed tissues have been shown to yield compromised RNA compared with that obtained from frozen tissue. To begin to assess the performance of RNA extracted from FFPE samples on a microarray format, we compared RNA from a model system of pelleted lipopolysaccharidestimulated human bone marrow stromal cells that were snap frozen with RNA from FFPE cells. RNA integrity and Affymetrix quality control parameters were assessed, and differentially regulated genes were analyzed with Ingenuity Pathway Analysis software. Results demonstrate that both snap-frozen and FFPE samples yielded intact RNA suitable for amplification prior to Affymetrix GeneChip analysis. Although some transcriptional information was lost with RNA extracted from the FFPE samples, Ingenuity Pathway Analysis revealed that the major pathways identified as affected by drug treatment were similar. Results show that FFPE samples are amenable to Affymetrix GeneChip analysis, expanding the possibility for expression profiling on archived tissue blocks in pathology laboratories. (J Histochem Cytochem 54:1229-1237, 2006)

Activation of peroxisome proliferator-activated receptor-alpha protects the heart from ischemia/reperfusion injury.

Background—Peroxisome proliferator–activated receptor-&agr; (PPAR-&agr;) is expressed in the heart and regulates genes involved in myocardial fatty acid oxidation (FAO). The role of PPAR-&agr; in acute ischemia/reperfusion myocardial injury remains unclear. Methods and Results—The coronary arteries of male mice were ligated for 30 minutes. After reperfusion for 24 hours, ischemic and infarct sizes were determined. A highly selective and potent PPAR-&agr; agonist, GW7647, was administered by mouth for 2 days, and the third dose was given 1 hour before ischemia. GW7647 at 1 and 3 mg · kg−1 · d−1 reduced infarct size by 28% and 35%, respectively (P <0.01), and myocardial contractile dysfunction was also improved. Cardioprotection by GW7647 was completely abolished in PPAR-&agr;–null mice. Ischemia/reperfusion downregulated mRNA expression of cardiac PPAR-&agr; and FAO enzyme genes, decreased myocardial FAO enzyme activity and in vivo cardiac fat oxidation, and increased serum levels of free fatty acids. All of these changes were reversed by GW7647. Moreover, GW7647 attenuated ischemia/reperfusion-induced release of multiple proinflammatory cytokines and inhibited neutrophil accumulation and myocardial expression of matrix metalloproteinases-9 and -2. Furthermore, GW7647 inhibited nuclear factor-&kgr;B activation in the heart, accompanied by enhanced levels of inhibitor-&kgr;B&agr;. Conclusions—Activation of PPAR-&agr; protected the heart from reperfusion injury. This cardioprotection might be mediated through metabolic and antiinflammatory mechanisms. This novel effect of the PPAR-&agr; agonist could provide an added benefit to patients treated with PPAR-&agr; activators for dyslipidemia.

p38 MAPK Inhibition Reduces Aortic Ultrasmall Superparamagnetic Iron Oxide Uptake in a Mouse Model of Atherosclerosis. MRI Assessment

Objective—Ultrasmall superparamagnetic iron oxide (USPIO) contrast agents have been used for noninvasive MRI assessment of atherosclerotic plaque inflammation. The purpose of this study was to noninvasively evaluate USPIO uptake in aorta of apoE−/− mice and to determine the effects of Angiotensin II (Ang II) infusion and chronic antiinflammatory treatment with a p38 MAPK inhibitor on this uptake. Methods and Results—ApoE−/− mice were administered saline or Ang II (1.44 mg/kg/d) for 21 days. In vivo MRI assessment of USPIO uptake in the aortic arch was observed in all animals. However, although the Ang II group had significantly higher absolute iron content (↑103%, P<0.001) in the aortic arch compared with the saline group, the p38 MAPK inhibitor (SB-239063, 150 mg/kg/d) treatment group did not (↑6%, NS). The in vivo MRI signal intensity was significantly correlated to the absolute iron content in the aortic arch. Histological evaluation of the aortic root lesion area showed colocalization of USPIO with macrophages and a reduction in USPIO but not macrophage content with SB-239063 treatment. Conclusion—The present study demonstrates that noninvasive assessment of USPIO uptake, as a marker for inflammation in murine atherosclerotic plaque, is feasible and that p38 MAPK inhibition attenuates the uptake of USPIO in aorta of Ang II–infused apoE−/− mice.

Protein Extraction of Formalin-fixed, Paraffin-embedded Tissue Enables Robust Proteomic Profiles by Mass Spectrometry

Global mass spectrometry (MS) profiling and spectral count quantitation are used to identify unique or differentially expressed proteins and can help identify potential biomarkers. MS has rarely been conducted in retrospective studies, because historically, available samples for protein analyses were limited to formalin-fixed, paraffin-embedded (FFPE) archived tissue specimens. Reliable methods for obtaining proteomic profiles from FFPE samples are needed. Proteomic analysis of these samples has been confounded by formalin-induced protein cross-linking. The performance of extracted proteins in a liquid chromatography tandem MS format from FFPE samples and extracts from whole and laser capture microdissected (LCM) FFPE and frozen/optimal cutting temperature (OCT)- embedded matched control rat liver samples were compared. Extracts from FFPE and frozen/OCT-embedded livers from atorvastatin-treated rats were further compared to assess the performance of FFPE samples in identifying atorvastatin-regulated proteins. Comparable molecular mass representation was found in extracts from FFPE and OCT-frozen tissue sections, whereas protein yields were slightly less for the FFPE sample. The numbers of shared proteins identified indicated that robust proteomic representation from FFPE tissue and LCM did not negatively affect the number of identified proteins from either OCT-frozen or FFPE samples. Subcellular representation in FFPE samples was similar to OCT-frozen, with predominantly cytoplasmic proteins identified. Biologically relevant protein changes were detected in atorvastatin-treated FFPE liver samples, and selected atorvastatin-related proteins identified by MS were confirmed by Western blot analysis. These findings demonstrate that formalin fixation, paraffin processing, and LCM do not negatively impact protein quality and quantity as determined by MS and that FFPE samples are amenable to global proteomic analysis.

Effects of Food Restriction on Testis and Accessory Sex Glands in Maturing Rats

Reduced food consumption and associated lower body weights may occur in subacute toxicity studies. The short-term effects of food restriction (FR) on body and reproductive organ weights, hormones, and testis histology were assessed in Sprague-Dawley rats fed 20% to 36% less (21 g feed/day) than rats fed ad libitum (AL) starting at six, eight, ten, or twelve weeks of age for two or six weeks. Body weight and relative seminal vesicle, ventral prostate, and/or epididymis weights were reduced in rats FR for two or six weeks. Degeneration of stage VII pachytene spermatocytes was seen in rats FR for six weeks when initiated at eight, ten, and twelve weeks of age. Plasma testosterone concentrations were lower in rats FR at ages six to eight weeks, eight to ten weeks, six to twelve weeks, and eight to fourteen weeks. Luteinizing hormone was not statistically different in FR rats compared with AL counterparts. Therefore, duration of lower food intake had a greater impact on spermatogenesis, whereas a younger initial age of lower food intake was more influential on testosterone levels. These interactions are important in the interpretation of subacute toxicology studies employing FR or when test articles lower food consumption relative to AL-fed rats.

Inhibition of ALK5 Signaling Induces Physeal Dysplasia in Rats

TGF-|β|, and its type 1 (ALK5) receptor, are critical to the pathogenesis of fibrosis. In toxicologic studies of 4 or more days in 10-week-old Sprague–Dawley rats, using an ALK5 inhibitor (GW788388), expansion of hypertrophic and proliferation zones of femoral physes were noted. Subphyseal hyperostosis, chondrocyte hypertrophy/hyperplasia, and increased matrix were present. Physeal zones were laser microdissected from ALK5 inhibitor-treated and control rats sacrificed after 3 days of treatment. Transcripts for TGF-|β|1, TGF-|β|2, ALK5, IHH, VEGF, BMP-7, IGF-1, bFGF, and PTHrP were amplified by real-time PCR. IGF and IHH increased in all physis zones with treatment, but were most prominent in prehypertrophic zones. TGF-|β|2, bFGF and BMP7 expression increased in proliferative, pre- and hypertrophic zones. PTHrP expression was elevated in proliferative zones but decreased in hypertrophic zones. VEGF expression was increased after treatment in pre- and hypertrophic zones. ALK5 expression was elevated in prehypertrophic zones. Zymography demonstrated gelatinolytic activity was reduced after treatment. Apoptotic markers (TUNEL and caspase-3) were decreased in hypertrophic zones. Proliferation assessed by Topoisomerase II and Ki67 was increased in multiple zones. Movat stains demonstrated that proteoglycan deposition was altered. Physeal changes occurred at doses well above those resulting in fibrosis. Interactions of factors is important in producing the physeal dysplasia phenotype.

Transcriptional Profiling of Laser Capture Microdissected Rat Arterial Elements: Fenoldopam-induced Vascular Toxicity as a Model System:

Transcriptional profiling of specific elements of vasculature from animal models of vascular toxicity is an approach to gain insight into molecular mechanisms of vascular injury. Feasibility of using laser capture microdissection (LCM) to evaluate differential gene expression in selected elements of mesenteric arteries (MA) from untreated rats and rats given a single vasotoxic dose of 100 mg/kg Fenoldopam and euthanized 1 or 4 hours postdose was assessed. Regions of MA (endothelial cells [EC] and vascular smooth muscle cells [VSMC]) were selectively microdissected from optimal-cutting-temperature (O.C.T.)-embedded-frozen tissue sections. RNA was isolated, linearly amplified (LA), and hybridized to Affymetrix GeneChips®. Enrichment for specific vascular elements was evident by unique gene-expression profiles. Statistical analysis indicated that Fenoldopam treatment resulted in differential expression of 333 versus 458 genes in EC and 371 versus 618 genes in VSMC at the 1-hour or 4-hour time point, respectively. Analysis of regulated EC and VSMC genes common to both time points identified several gene functions or pathways affected by treatment. Several genes were identified in EC and/or VSMC that have not been previously linked to vascular structure or function. These data indicate that tissue–element-enrichment by LCM in conjunction with LA and GeneChip analysis offers a refined approach for assessment of injury-mediated transcriptome changes in distinct elements of the vasculature.

Choice of Morphometric Methods and Consequences in the Regulatory Environment

In certain cases, quantitative tissue structural data derived from tissue sections may be required to make critical decisions in the drug development or risk assessment process. Most frequently, these questions center on test article–related effects on cell number. In this opinion article, the limitations of estimating cell number by standard cell or nuclear profile counts from sections/blocks collected for routine histopathology are discussed from both a scientific and regulatory perspective and contrasted with the robust, sensitive, statistically based methods of design-based stereology. Specific existing industry practices are reviewed. Recent advances in stereological theory, software, hardware, and automated immunohistochemical staining now make it feasible to implement unbiased stereological methods to assess test article–related effects on cell number in a regulatory toxicology setting. These design-based stereological methods for counting cells are recommended when the quantification of small changes in cell number is critical to the risk assessment or decision-making process. These methods provide levels of sensitivity and statistical guarantees of accuracy that no other currently available tissue section–based methodology can provide.

Topic of Histopathology Blinding in Nonclinical Safety Biomarker Qualification Studies

Various consortia and working groups, composed of professionals from industry, academia, and government institutions, have undertaken or are undertaking nonclinical work to qualify safety biomarkers of tissue injury and function. As this work has developed and voluntary data have been submitted to regulatory authorities, study practices have come under close scrutiny in an attempt to ensure that the best science is consistently being applied. One practice that has been discussed in a variety of venues is the process used to generate histopathology data as additional end points and/or correlates in these studies. Histopathologic evaluation plays a critical role in these biomarker studies, because microscopic demonstration of given cellular processes are commonly used as a reference standard to assess diagnostic performance of candidate new biomarkers using methods such as receiver operating characteristic (ROC) analyses. In this regard, it is acknowledged that pathologists should not perform the histopathologic evaluation with knowledge of the candidate biomarker data; however, it has been questioned whether pathologists should conduct the histopathology evaluation for these biomarker studies without knowledge of treatment or other study-related data (i.e., ‘‘blinded’’ evaluation). Research pathologists, and especially the Society of Toxicologic Pathology (STP), have closely examined regulatory study histopathology practices (Crissman et al. 2004; Wandall, Hansson and Ruden 2007). Recently, a set of Best Practices for the conduct of histopathology review within nonclinical safety studies was endorsed by the STP (Crissman et al. 2004). Key elements of these Best Practices indicate that the study pathologist should be informed as follows: (1) have knowledge of the treatment group from which the sample was obtained; and (2) have complete knowledge of all available study-related data that are associated with the animal from which the tissue was obtained. As these practices have been employed successfully for decades in the context of regulatory toxicology studies designed to assess human safety of new chemical entities with complete regulatory acceptance, the rationale for following a different process for biomarker validation and qualification is unclear to these authors. This informed type of analysis (described above) is often referred to as ‘‘unblinded,’’ because the study pathologist has knowledge of dose groups and other study-related data at the time that the review is being conducted. It is considered that ‘‘unblinded’’ evaluation is critical to discriminate treatment-related changes from background, especially when subtle treatment-related effects increase the incidence or severity of spontaneous background findings. As needed, the study pathologist may reexamine tissues using a procedure sometimes termed ‘‘targeted masked’’ evaluation. This procedure entails reexamination of selected or all treated dose groups, randomly combined with controls and without knowledge of animal or group identity, to determine whether a subtle or equivocal finding can be identified consistently from control tissues. This second evaluation is performed after the pathologist feels confident that each finding has been fully characterized, to ensure either that subtle findings are assessed for incidence and severity or that equivocal findings are assessed to discriminate a true change from spontaneous background (in an unbiased manner). Further to the nonclinical safety study Best Practices endorsed by STP, a peer-review process is generally used as a method for quality assurance, in which a second pathologist can corroborate the interpretations of the study pathologist. This second evaluation is typically conducted in an ‘‘unblinded’’ fashion, as this evaluation is not meant to generate new data but rather to provide a second party evaluation of the interpretations and conclusions of the primary pathologist. Taking into consideration all available historical data as well as current practices of histopathology data generation, the current authors were tasked by the Scientific and Regulatory Policy Committee of the STP to consider the development of either a Best Practices or a Points to Consider guideline for industry and regulators, with emphasis on histopathology practices related to biomarker qualification. Although the final document will not be available until later, we wished to present our preliminary opinion based on the aforementioned Best Practices (Crissman et al. 2004). The present authors recognize that histopathology is a special discipline in which considerations for best practices may differ from those of other disciplines used in biomarker evaluation. The present authors also acknowledge that some toxicities are either unique to humans or idiosyncratic in nature and thus undetectable in standard nonclinical studies. Therefore, the method of evaluating slides would not affect the sensitivity for detection of such changes. Conversely, the present authors agree that ‘‘unblinded’’ slide evaluation creates a high degree of fidelity and consistency in the ability to identify animal toxicities. We concur with