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Increased osteopontin (OPN) expression associates with increased myocyte apoptosis and myocardial dysfunction. The objective of this study was to identify the receptor for OPN and get insight into the mechanism by which OPN induces cardiac myocyte apoptosis. Adult rat ventricular myocytes (ARVMs) and transgenic mice expressing OPN in a myocyte-specific manner were used for in vitro and in vivo studies. Treatment with purified OPN (20 nM) protein or adenoviral-mediated OPN expression induced apoptosis in ARVMs. OPN co-immunoprecipitated with CD44 receptors, not with β1 or β3 integrins. Proximity ligation assay confirmed interaction of OPN with CD44 receptors. Neutralizing anti-CD44 antibodies inhibited OPN-stimulated apoptosis. OPN activated JNKs and increased expression of Bax and levels of cytosolic cytochrome c, suggesting involvement of mitochondrial death pathway. OPN increased endoplasmic reticulum (ER) stress, as evidenced by increased expression of Gadd153 and activation of caspase-12. Inhibition of JNKs using SP600125 or ER stress using salubrinal or caspase-12 inhibitor significantly reduced OPN-stimulated apoptosis. Expression of OPN in adult mouse heart in myocyte-specific manner associated with decreased left ventricular function and increased myocyte apoptosis. In the heart, OPN expression increased JNKs and caspase-12 activities, and expression of Bax and Gadd153. Thus, OPN, acting via CD44 receptors, induces apoptosis in myocytes via the involvement of mitochondrial death pathway and ER stress.

Objective— HSPA12B is the newest member of HSP70 family of proteins and is enriched in atherosclerotic lesions. This study focused on HSPA12B expression in mice and its involvement in angiogenesis. Methods and Results— The expression of HSPA12B in mice and cultured cells was studied by: (1) Northern blot; (2) in situ hybridization; (3) immunostaining with HSPA12B-specific antibodies; and (4) expressing Enhanced-Green-Fluorescent-Protein under the control of the HSPA12B promoter in mice. The function of HSPA12B was probed by an in vitro angiogenesis assay (Matrigel) and a migration assay. Interacting proteins were identified through a yeast two-hybrid screening. HSPA12B is predominantly expressed in vascular endothelium and induced during angiogenesis. In vitro angiogenesis and migration are inhibited in human umbilical vein endothelial cells in the presence of HSPA12B-neutralizing antibodies. HSPA12B interacts with multiple proteins in yeast 2-hybrid system. Conclusions— We provide the first evidence to our knowledge that the HSPA12B is predominantly expressed in endothelial cells, required for angiogenesis, and interacts with known angiogenesis regulators. We postulate that HSPA12B provides a new mode of angiogenesis regulation and a novel therapeutic target for angiogenesis-related diseases.

Extracellular Ub is an immune modulator that plays a role in suppression of inflammation, organ injury, myocyte apoptosis, and fibrosis. The purpose of this study was to investigate the effects of extracellular Ub on the process of cardiac angiogenesis. CMECs and aortic tissue were isolated from rats to measure changes in angiogenic protein levels and to assess angiogenic responses to extracellular Ub. In CMECs, extracellular Ub increased protein levels of VEGF-A and MMP-2, known angiogenesis regulators. CMECs demonstrated enhanced rearrangement of fibrillar actin and migration in response to Ub treatment. Ub-treated CMECs demonstrated an increase in tube network formation which was inhibited by the CXCR4 receptor antagonist, AMD3100. Methylated Ub, unable to form polyubiquitin chains, enhanced tube network formation. Aortic ring sprouting assays demonstrated that Ub increases microvessel sprouting in the Matrigel. The results of our study suggest a novel role for extracellular Ub in cardiac angiogenesis, providing evidence that extracellular Ub, at least in part acting via the CXCR4 receptor, has the potential to facilitate the process of angiogenesis in myocardial endothelial cells.

Sjogren's syndrome is an extremely complex and currently incurable autoimmune disorder, which occurs primarily in females, and is associated with lacrimal gland inflammation, meibomian gland dysfunction, and severe dry eye. We hypothesize that androgen deficiency, which reportedly occurs in primary and secondary Sjogren's syndrome (e.g., systemic lupus erythematosus, rheumatoid arthritis), is a critical etiologic factor in the pathogenesis of dry eye syndromes. We further hypothesize that androgen treatment to the ocular surface will promote both lacrimal and meibomian gland function and alleviate both “aqueous-deficient” and “evaporative” dry eye. Our results demonstrate that androgens regulate both lacrimal and meibomian gland function, and suggest that topical androgen administration may serve as a safe and effective therapy for the treatment of dry eye in Sjogren's syndrome.

Spinal cord stimulation (SCS) inhibits substance P (SP) release and decreases the expression of the transient receptor potential vanilloid 1 (TRPV1) in the spinal cord at thoracic 4 (T4) during cardiac ischemia in rat models (Ding et al., 2007). We hypothesized that activation of TRPV1 in the T4 spinal cord segment by intermittent occlusion of the left anterior descending coronary artery (CoAO) mediates spinal cord SP release. Experiments were conducted in urethane-anesthetized Sprague-Dawley male rats using SP antibody-coated microprobes to measure SP release at the central terminal endings of cardiac ischemic-sensitive afferent neurons (CISAN) in the spinal T4 dorsal horns. Vehicle, capsaicin (CAP; TRPV1 agonist) and capsazepine (CZP; TRPV1 antagonist) were injected into the left T4 prior to stimulation of CISAN by intermittent CoAO (with or without upper cervical SCS). CAP induced endogenous SP release from laminae I and II in the T4 spinal cord above baseline. Conversely, CZP injections significantly inhibited SP release from laminae I-VII in the T4 spinal cord segment below baseline. CZP also attenuated CoAO-induced SP release, while T4 injections of CZP with SCS completely restored SP release to basal levels during CoAO activation. CAP increased the number of c-Fos (a marker for CISAN activation) positive T4 dorsal horn neurons compared to sham-operated animals, while CZP (alone or during CoAO and SCS+CoAO) significantly reduced the number of c-Fos positive neurons. These results suggest that spinal release of the putative nociceptive transmitter SP occurs, at least in part, via a TRPV1 mechanism.

Antibody-coated microprobes have been demonstrated to be useful for detecting the release of neuropeptide transmitters from discrete sites in the central nervous system (CNS). This technique uses glass micropipettes taken through a series of chemical coatings, starting with a γ-aminopropyltriethoxysilane solution and ending with the antibody specific to the peptide transmitter of interest. The key to the reliability and repeatability of the technique is a uniform, even coating of the siloxane polymer to the glass micropipette. The microprobes, as they are called following the completion of the coating process, are inserted stereotaxically into a specific area of the CNS and the physiological intervention is performed. Tip diameters are around 5-10 μm and, depending on the length of the pipette inserted into the CNS, diameters of the pipette shaft will approach 40-50 μm. Once removed, the microprobe is then incubated with the radiolabeled peptide. Binding of the radiolabeled peptide will occur to the antibody sites not occupied by the endogenously released peptide. The images of the microprobes on sensitive autoradiographic film are analyzed for differences in the optical density along a specified length of probe. Areas of lighter density signify sites along the microprobe where endogenous peptide was biologically released during the physiological intervention. Knowing the exact location of the probe tip in vivo in the CNS permits identification of neurophysiological sites corresponding along the length of the microprobe where the peptide was released.

We have previously shown that HspA12B, a member of HspA70 family subfamily 12, is a novel angiogenesis regulator that is preferentially expressed in endothelial cells (ECs) and required for angiogenesis in vitro . The mechanism by which HspA12B regulates angiogenesis, however, is unknown. In this study we identified AKAP12/SSeCKS as a HSPA12B-interacting protein through a yeast two-hybrid screening and confirmed the interaction by co-immunoprecipitation and co-localization. We observed that HspA12B negatively regulated the expression of AKAP12/SSeCKS, a cancer metastasis repressor that inhibits VEGF expression and angiogen-esis. In HUVEC, HspA12B knockdown increased AKAP12 levels, decreased VEGF by more than 75%, and down-regulated Akt and pAkt; whereas HspA12B over expression decreased AKAP12 and more than doubled VEGF levels. We further identified a 32-AA domain in AKAP12 that was capable of interacting with HspA12B. Overexpression of this 32-AA domain in HUVEC disrupted the HspA12B-AKAP12 interaction and decreased VEGF expression by more than 70%, suggesting the importance of HspA12B-AKAP12 interaction in regulating VEGF. We also observed that HspA12B expression was increased more than 2 folds in ECs by hypoxia or shearing stress, and induced in ischemic rat heart. Inhibition of HspA12B abolished hypoxia-induced tubule formation. Adeno-HspA12B promoted angiogenesis in DIVAA assay. We concluded that this is the first evidence that HspA12B promotes angiogenesis through regulating VEGF by way of suppressing AKAP12. Our finding is the first example of an EC-specific molecular chaperone acting as the regulator of angiogenesis.

Significant, sex-associated differences exist in the physiology and pathophysiology of the lacrimal gland. We hypothesize that many of these differences are due to fundamental variations in gene expression. The purpose of this study was to determine the extent to which sex-related differences in gene expression are present in the lacrimal gland. Lacrimal glands were obtained from adult male and female BALB/c mice (n=5-10mice/sex/experiment), pooled according to sex and processed for the isolation of RNA. Samples were analyzed for differentially expressed mRNAs by using Atlas Mouse cDNA Expression Arrays, cDNA amplification techniques, GEM 1 and 2 gene chips, CodeLink bioarrays and quantitative real-time PCR (qPCR) procedures. Quantitative evaluation of Atlas Array gene expression was performed with an image analysis system developed in our laboratory, whereas gene chip data were analyzed with Rosetta Resolver and GeneSifter.Net software. Statistical significance was determined by using Student's t-test. Our results with CodeLink bioarrays show that sex has a significant influence on the expression of over 490 genes in the mouse lacrimal gland. These genes are involved in a wide range of biological processes, molecular functions and cellular components, including such activities as development, growth, transcription, metabolism, signal transduction, transport, receptor activity and protein and nucleic acid binding. The expression of selected genes was confirmed by the use of GEM gene chips and qPCR. Our findings also demonstrate that certain methodological approaches are less useful in attempting to assess the magnitude of sex-associated differences in the lacrimal gland. These results support our hypothesis that sex-related differences in gene expression play a role in the sexual dimorphism of the lacrimal gland.

Our recent research has led to our hypothesis that gender and sex steroid hormones are critical factors in the regulation of the meibomian gland and pathogenesis of evaporative dry eye. The objective of this article is to briefly summarize the experimental basis for this hypothesis.

Meibomian gland function is extremely important in promoting the health and integrity of the ocular surface.1–5 This gland, through its synthesis and secretion of lipids, enhances the stability and prevents the evaporation of the tear film.1–5 Meibomian gland dysfunction, in turn, leads to lipid insufficiency, tear film instability and evaporative dry eye.1–5 In fact, meibomian gland dysfunction is thought to be the primary cause of dry eye syndromes throughout the world.6 However, despite the importance of the meibomian gland in maintaining the well-being of the eye, very little research has been published concerning the physiological regulation of this tissue.

Previous research has shown that a sex-related difference exists in the casual level of meibomian gland lipids on the lid margin, with women having lower amounts than men from puberty to menopause.1 We hypothesize that this difference may possibly reflect a gender-associated variation in gene expression, and consequent function, of the meibomian gland. Moreover, we hypothesize that this sex-related difference may relate in part to the influence of androgens, which are known to modulate the meibomian gland.2–9 The purpose of this study was to begin to test these hypotheses.