Linda J. Lowe-Krentz

Identification of 1- and 3-methylhistidine as biomarkers of skeletal muscle toxicity by nuclear magnetic resonance-based metabolic profiling

Nuclear magnetic resonance (NMR)-based metabolomic profiling identified urinary 1- and 3-methylhistidine (1- and 3-MH) as potential biomarkers of skeletal muscle toxicity in Sprague-Dawley rats following 7 and 14 daily doses of 0.5 or 1mg/kg cerivastatin. These metabolites were highly correlated to sex-, dose- and time-dependent development of cerivastatin-induced myotoxicity. Subsequently, the distribution and concentration of 1- and 3-MH were quantified in 18 tissues by gas chromatography-mass spectrometry. The methylhistidine isomers were most abundant in skeletal muscle with no fiber or sex differences observed; however, 3-MH was also present in cardiac and smooth muscle. In a second study, rats receiving 14 daily doses of 1mg/kg cerivastatin (a myotoxic dose) had 6- and 2-fold elevations in 1- and 3-MH in urine and had 11- and 3-fold increases in 1- and 3-MH in serum, respectively. Selectivity of these potential biomarkers was tested by dosing rats with the cardiotoxicant isoproterenol (0.5mg/kg), and a 2-fold decrease in urinary 1- and 3-MH was observed and attributed to the anabolic effect on skeletal muscle. These findings indicate that 1- and 3-MH may be useful urine and serum biomarkers of drug-induced skeletal muscle toxicity and hypertrophy in the rat, and further investigation into their use and limitations is warranted.

Biomarkers of Drug-Induced Skeletal Muscle Injury in the Rat: Troponin I and Myoglobin

The purpose of this investigation was to determine the utility of fast-twitch skeletal muscle troponin I (fsTnI) and urinary myoglobin (uMB) as biomarkers of skeletal muscle injury in 8-week-old Sprague-Dawley rats. fsTnI and uMB were quantified by enzyme-linked immunosorbent assay and compared with standard clinical assays including creatine kinase, aldolase, aspartate aminotransferase, and histopathological assessments. Detectable levels of uMB were normalized to urinary creatinine to control for differences in renal function. Seven compounds, including those with toxic effects on skeletal muscle, cardiac muscle, or liver, were evaluated. fsTnI was typically nondetectable (< 5.9 ng/ml serum) in vehicle-treated female and male rats but increased in a dose-dependent manner to at least 300 ng/ml in cerivastatin-induced severe fast-twitch specific myotoxicity. Minimal myopathy induced by investigational compounds BMS-600149 and BMS-687453 increased serum fsTnI to about 30-50 ng/ml, suggesting a reasonable dynamic range for detecting mild to severe skeletal muscle toxicity. In direct contrast, fsTnI was only marginally increased relative to population control values in rats treated with triamcinolone acetonide, which produces muscle atrophy or the cardiotoxins isoproterenol and CoCl2. uMB was typically nondetectable (< 1.6 ng/ml urine) in vehicle-treated female and male rats but increased to approximately 140, 300, and 30 ng/mg creatinine in rats treated with cerivastatin, BMS-687453, and triamcinolone acetonide, respectively. Cardiotoxicity also increased uMB in rats treated with isoproterenol and CoCl2 with urine concentrations ranging from 20 to 30 ng/mg creatinine. Severe hepatotoxicity (coumarin) did not significantly affect serum fsTnI or uMB levels. Collectively, these data suggest that fsTnI is specific for skeletal muscle toxicity, whereas uMB is nonspecific, increasing with skeletal muscle and cardiac toxicity. Accordingly, the complement of fsTnI and uMB, in conjunction with standard clinical assays may comprise a useful diagnostic panel for assessing drug-induced myopathy in rats.

Antibodies against a putative heparin receptor slow cell proliferation and decrease MAPK activation in vascular smooth muscle cells.

Heparin has long been known to slow the growth of vascular smooth muscle cells. However, the mechanism(s) by which heparin acts has yet to be resolved. The identification of a putative heparin receptor in endothelial cells with antibodies that blocked heparin binding to the cells provided the means to further examine the possible involvement of a heparin receptor in smooth muscle cell responses to heparin. Immunoprecipitation of a smooth muscle cell protein with the anti‐heparin receptor antibodies provided evidence that the protein was present in smooth muscle cells. Experiments with the anti‐heparin receptor antibodies indicate that the antibodies can mimic heparin in decreasing PDGF induced thymidine and BrdU incorporation. The anti‐heparin receptor antibodies were also found to decrease MAPK activity levels after activation similarly to heparin. These results support the identification of a heparin receptor and its role in heparin effects on vascular smooth muscle cell growth.

Roles of mouse sperm-associated alpha-L-fucosidases in fertilization

Sperm‐associated α‐L‐fucosidases have been implicated in fertilization in many species. Previously, we documented the existence of α‐L‐fucosidase in mouse cauda epididymal contents, and showed that sperm‐associated α‐L‐fucosidase is cryptically stored within the acrosome and reappears within the sperm equatorial segment after the acrosome reaction. The enrichment of sperm membrane‐associated α‐L‐fucosidase within the equatorial segment of acrosome‐reacted cells implicates its roles during fertilization. Here, we document the absence of α‐L‐fucosidase in mouse oocytes and early embryos, and define roles of sperm associated α‐L‐fucosidase in fertilization using specific inhibitors and competitors. Mouse sperm were pretreated with deoxyfuconojirimycin (DFJ, an inhibitor of α‐L‐fucosidase) or with anti‐fucosidase antibody; alternatively, mouse oocytes were pretreated with purified human liver α‐L‐fucosidase. Five‐millimolar DFJ did not inhibit sperm–zona pellucida (ZP) binding, membrane binding, or fusion and penetration, but anti‐fucosidase antibody and purified human liver α‐L‐fucosidase significantly decreased the frequency of these events. To evaluate sperm‐associated α‐L‐fucosidase enzyme activity in post‐fusion events, DFJ‐pretreated sperm were microinjected into oocytes, and 2‐pronuclear (2‐PN) embryos were treated with 5 mM DFJ with no significant effects, suggesting that α‐L‐fucosidase enzyme activity does not play a role in post‐fusion events and/or early embryo development in mice. The recognition and binding of mouse sperm to the ZP and oolemma involves the glycoprotein structure of α‐L‐fucosidase, but not its catalytic action. These observations suggest that deficits in fucosidase protein and/or the presence of anti‐fucosidase antibody may be responsible for some types of infertility. Mol. Reprod. Dev. 80: 273–285, 2013.

Fluid shear stress-induced JNK activity leads to actin remodeling for cell alignment

Fluid shear stress (FSS) exerted on endothelial cell (EC) surfaces induces actin cytoskeleton remodeling through mechanotransduction. This study was designed to determine whether FSS activates Jun N‐terminal kinase (JNK), to examine the spatial and temporal distribution of active JNK relative to the actin cytoskeleton in ECs exposed to different FSS conditions, and to evaluate the effects of active JNK on actin realignment. Exposure to 15 and 20 dyn/cm2 FSS induced higher activity levels of JNK than the lower 2 and 4 dyn/cm2 flow conditions. At the higher FSS treatments, JNK activity increased with increasing exposure time, peaking 30 min after flow onset with an eightfold activity increase compared to cells in static culture. FSS‐induced phospho‐JNK co‐localized with actin filaments at cell peripheries, as well as with stress fibers. Pharmacologically blocking JNK activity altered FSS‐induced actin structure and distribution as a response to FSS. Our results indicate that FSS‐induced actin remodeling occurs in three phases, and that JNK plays a role in at least one, suggesting that this kinase activity is involved in mechanotransduction from the apical surface to the actin cytoskeleton in ECs. J. Cell. Physiol. 226: 110–121, 2010.

Actin realignment and cofilin regulation are essential for barrier integrity during shear stress

Vascular endothelial cells and their actin microfilaments align in the direction of fluid shear stress (FSS) in vitro and in vivo. To determine whether cofilin, an actin severing protein, is required in this process, the levels of phospho‐cofilin (serine‐3) were evaluated in cells exposed to FSS. Phospho‐cofilin levels decreased in the cytoplasm and increased in the nucleus during FSS exposure. This was accompanied by increased nuclear staining for activated LIMK, a cofilin kinase. Blocking stress kinases JNK and p38, known to play roles in actin realignment during FSS, decreased cofilin phosphorylation under static conditions, and JNK inhibition also resulted in decreased phospho‐cofilin during FSS exposure. Inhibition of dynamic changes in cofilin phosphorylation through cofilin mutants decreased correct actin realignment. The mutants also decreased barrier integrity as did inhibition of the stress kinases. These results identify the importance of cofilin in the process of actin alignment and the requirement for actin realignment in endothelial barrier integrity during FSS. J. Cell. Biochem. 114: 782–795, 2013.

Active Stress Kinases in Proliferating Endothelial Cells Associated with Cytoskeletal Structures

It has become increasingly clear that stress-activated protein kinases have cytoplasmic substrates in addition to well-established transcription factor substrates in cell nuclei. The present study documented specific cytoplasmic locations of these enzymes in proliferating vascular cells. Immunofluorescent staining for active c-jun NH2-terminal kinase (JNK), the precipitation of JNK with microfilaments, and the loss of fiber-associated active JNK after cytochalasin treatment, but not nocodazole treatment, together indicate that active JNK is associated with stress fibers. The lack of complete scaffold colocalization and the total lack of immediate upsteam kinase colocalization along with the inability of JNK inhibitors to alter JNK-microfilament associations suggest that the microfilament association is not simply involved in enzyme activation. In addition, active p38 was found along with vinculin in focal adhesions. Although the p38 in focal adhesions could also be disrupted by cytochalasin treatment, it remained stable after nocodazole treatment. These results support the hypothesis that vascular cell stress kinase enzymes are important for signal transduction in the cytoplasm. The localization of active stress-activated protein kinases to specific cytoskeletal structures in proliferating cells suggests that subsets of these enzymes are involved in signal transduction to and/or from the cytoskeleton under conditions that include vascular cell proliferation.

Heparin responses in vascular smooth muscle cells involve cGMP-dependent protein kinase (PKG).

Published data provide strong evidence that heparin treatment of proliferating vascular smooth muscle cells results in decreased signaling through the ERK pathway and decreases in cell proliferation. In addition, these changes have been shown to be mimicked by antibodies that block heparin binding to the cell surface. Here, we provide evidence that the activity of protein kinase G is required for these heparin effects. Specifically, a chemical inhibitor of protein kinase G, Rp‐8‐pCPT‐cGMS, eliminates heparin and anti‐heparin receptor antibody effects on bromodeoxyuridine incorporation into growth factor‐stimulated cells. In addition, protein kinase G inhibitors decrease heparin effects on ERK activity, phosphorylation of the transcription factor Elk‐1, and heparin‐induced MKP‐1 synthesis. Although transient, the levels of cGMP increase in heparin treated cells. Finally, knock down of protein kinase G also significantly decreases heparin effects in growth factor‐activated vascular smooth muscle cells. Together, these data indicate that heparin effects on vascular smooth muscle cell proliferation depend, at least in part, on signaling through protein kinase G. J. Cell. Physiol. 229: 2142–2152, 2014.

Biomimetic channel modeling local vascular dynamics of pro-inflammatory endothelial changes

Endothelial cells form the inner lining of blood vessels and are exposed to various factors like hemodynamic conditions (shear stress, laminar, and turbulent flow), biochemical signals (cytokines), and communication with other cell types (smooth muscle cells, monocytes, platelets, etc.). Blood vessel functions are regulated by interactions among these factors. The occurrence of a pathological condition would lead to localized upregulation of cell adhesion molecules on the endothelial lining of the blood vessel. This process is promoted by circulating cytokines such as tumor necrosis factor-alpha, which leads to expression of intercellular adhesion molecule-1 (ICAM-1) on the endothelial cell surface among other molecules. ICAM-1 is critical in regulating endothelial cell layer dynamic integrity and cytoskeletal remodeling and also mediates direct cell-cell interactions as part of inflammatory responses and wound healing. In this study, we developed a biomimetic blood vessel model by culturing confluent, flow aligned, endothelial cells in a microfluidic platform, and performed real time in situ characterization of flow mediated localized pro-inflammatory endothelial activation. The model mimics the physiological phenomenon of cytokine activation of endothelium from the tissue side and studies the heterogeneity in localized surface ICAM-1 expression and F-actin arrangement. Fluorescent antibody coated particles were used as imaging probes for identifying endothelial cell surface ICAM-1 expression. The binding properties of particles were evaluated under flow for two different particle sizes and antibody coating densities. This allowed the investigation of spatial resolution and accessibility of ICAM-1 molecules expressed on the endothelial cells, along with their sensitivity in receptor-ligand recognition and binding. This work has developed an in vitro blood vessel model that can integrate various heterogeneous factors to effectively mimic a complex endothelial microenvironment and can be potentially applied for relevant blood vessel mechanobiology studies.

Human sperm CRISP2 is released from the acrosome during the acrosome reaction and re-associates at the equatorial segment.

Sperm CRISP2 has been proposed to be involved in sperm–egg fusion. After the acrosome reaction, it appears at the equatorial segment (EqS) of human sperm; the mechanism underlying the appearance of CRISP2 at the EqS remains unknown, though. Here, we provide evidence showing the re‐association of sperm acrosomal CRISP2 at the EqS during the acrosome reaction. Results showed that F‐actin is not involved in the relocalization of CRISP2. We found that basic, but not acidic, conditions can solubilize CRISP2 from sperm cells, suggesting that CRISP2 is a component of the acrosome and that it is released from the acrosome during the acrosome reaction. Purified, biotinylated human sperm acrosomal CRISP2 binds to the EqS of acrosome‐reacted sperm in a dose‐dependent manner, revealing that CRISP2 detected at the EqS of acrosome‐reacted sperm comes from the population stored in the acrosome. The association of CRISP2 at the EqS is very strong, and does not depend on ionic interactions or intermolecular disulfide bonds. Interestingly, the restriction of CRISP2 at the EqS was diminished when EGTA was present in the media, indicating that Ca2+ is required for maintaining CRISP2 at the EqS. This study supports the possibility that CRISP2 may help modify the EqS membrane to make this domain fusion‐competent. Mol. Reprod. Dev. 80: 488–502, 2013.