Overexpression of SERCA2 ATPaase in vascular smooth muscle cells treated with oxidized low density lipoprotein

Oxidized low density lipoprotein (oxLDL) has been identified as a potentially important atherogenic factor. Atherosclerosis is characterized by the accumulation of lipid and calcium in the vascular wall. OxLDL plays a significant role in altering calcium homeostasis within different cell types. In our previous study, chronic treatment of vascular smooth muscle cells (VSMC) with oxLDL depressed Ca²⁺ _i homeostasis and altered two Ca²⁺ release mechanisms in these cells (IP₃ and ryanodine sensitive channels). The purpose of the present study was to further define the effects of chronic treatment with oxLDL on the smooth muscle sarcoplasmic reticulum (SR) Ca²⁺ pump. One of the primary Ca²⁺ uptake mechanisms in VSMC is through the SERCA2 ATPase calcium pump in the sarcoplasmic reticulum. VSMC were chronically treated with 0.005-0.1 mg/ml oxLDL for up to 6 days in culture. Cells treated with oxLDL showed a significant increase in the total SERCA2 ATPase content. These changes were observed on both Western blot and immunocytochemical analysis. This increase in SERCA2 ATPase is in striking contrast to a significant decrease in the density of IP₃ and ryanodine receptors in VSMC as the result of chronic treatment with oxLDL. This response may suggest a specific adaptive mechanism that the pump undergoes to attempt to maintain Ca²⁺ homeostasis in VSMC chronically exposed to atherogenic oxLDL.     

Hand‐held,clinical dual mode ultrasound ‐ photoacoustic imaging of rat urinary bladder and its applications

Urinary bladder imaging is critical to diagnose urinary tract disorders, and bladder cancer. There is a great need for safe, non‐invasive, and sensitive imaging technique which enables bladder imaging. Photoacoustic imaging is a rapidly growing imaging technique for various biological applications. It can be combined with clinical ultrasound imaging system for hand‐held, dual modal ultrasound‐photoacoustic real‐time imaging. Structural (bladder wall) and functional (accretion of nanoparticles) bladder imaging is shown here with combined ultrasound and photoacoustic imaging in rats. Photoacoustic images of bladder wall is shown using black ink as the contrast agent. Chicken tissues were stacked on the abdomen of the animal to demonstrate the feasibility of photoacoustic imaging till a depth of 2 cm. Also, the feasibility of photoacoustic imaging for a common bladder disorder, vesicoureteral reflux is studied using urinary tract mimicking phantom. It is also shown that a clinical ultrasound system can be used for photoacoustic imaging of non‐invasive clearance study of gold nanorods from circulation by monitoring the gradual accumulation of the gold nanorods in the bladder. The time taken for accumulation of nanorods in the bladder can be used as an indicator of the clearance rate of the nanoparticle circulation from the body.      

Defective phosphatidic acid-phospholipase C signaling in diabetic cardiomyopathy

The effects of exogenous phosphatidic acid (PA) on Ca2+ transients and contractile activity were studied in cardiomyocytes isolated from chronic streptozotocin-induced diabetic rats. In control cells, 25 microM PA induced a significant increase in active cell shortening and Ca2+ transients. PA increased IP3 generation in the control cardiomyocytes and its inotropic effects were blocked by a phospholipase C inhibitor. In cardiomyocytes from diabetic rats, PA induced a 25% decrease in active cell shortening and no significant effect on Ca2+ transients. Basal and PA-induced IP3 generation in diabetic rat cardiomyocytes was 3-fold lower as compared to control cells. Sarcolemmal membrane PLC activity was impaired. Insulin treatment of the diabetic animals resulted in a partial recovery of PA responses. Our results, therefore, identify an important defect in the PA-PLC signaling pathway in diabetic rat cardiomyocytes, which may have significant implications for heart dysfunction during diabetes.     

Brca1 Mutations Enhance Mouse Reproductive Functions by Increasing Responsiveness to Male-Derived Scent

We compared the gene expression profiles of ovarian granulosa cells harboring either mutant or wild type Brca1 to follow up on our earlier observation that absence of a functional Brca1 in these important regulators of menstrual/estrous cycle progression leads to prolongation of the pre-ovulatory phase of the estrous cycle and to increased basal levels of circulating estradiol. Here we show that ovarian granulosa cells from mice carrying a conditional Brca1 gene knockout express substantially higher levels of olfactory receptor mRNA than granulosa cells from wild type littermates. This led us to hypothesize that reproductive functions in mutant female mice might be more sensitive to male-derived scent than in wild type female mice. Indeed, it is well established that isolation from males leads to complete cessation of mouse estrous cycle activity while exposure to olfactory receptor ligands present in male urine leads to resumption of such activity. We found that Brca1 ^-/- female mice rendered anovulatory by unisexual isolation resumed ovulatory activity more rapidly than their wild type littermates when exposed to bedding from cages where males had been housed. The prime mediator of this increased responsiveness appears to be the ovary and not olfactory neurons. This conclusion is supported by the fact that wild type mice in which endogenous ovaries had been replaced by Brca1-deficient ovarian transplants responded to male-derived scent more robustly than mutant mice in which ovaries had been replaced by wild type ovarian transplants. Our findings not only have important implications for our understanding of the influence of olfactory signals on reproductive functions, but also provide insights into mechanisms whereby genetic risk factors for breast and extra uterine Müllerian carcinomas may influence menstrual activity in human, which is itself an independent risk factor for these cancers.     

Effect of dietary hempseed intake on cardiac ischemia-reperfusion injury

Polyunsaturated fatty acids (PUFAs) have significant, cardioprotective effects against ischemia. Hempseed contains a high proportion of the PUFAs linoleic acid (LA) and alpha-linolenic acid (ALA), which may have opposing effects on postischemic heart performance. There are no reported data concerning the cardiovascular effects of dietary hempseed intake. A group of 40 male Sprague-Dawley rats were distributed evenly into four groups that were fed for 12 wk a normal rat chow supplemented with hempseed (5% and 10%), palm oil (1%), or a 10% partially delipidated hempseed that served as a control. Plasma ALA and gamma-linolenic acid levels were significantly elevated in the rats that were fed a 5% or 10% hempseed-supplemented diet, but in heart tissue only ALA levels were significantly elevated in the rats fed these diets compared with control. After the dietary interventions were completed, postischemic heart performance was evaluated by measuring developed tension, resting tension, the rates of tension development and relaxation, and the number of extrasystoles. Hearts from rats fed a hempseed-supplemented diet exhibited significantly better postischemic recovery of maximal contractile function and enhanced rates of tension development and relaxation during reperfusion than hearts from the other groups. These hearts, however, were not protected from the occurrence of extrasystoles, nor were the increases in resting tension altered during ischemia or reperfusion as a function of any dietary intervention. Our data demonstrate that dietary hempseed can provide significant cardioprotective effects during postischemic reperfusion. This appears to be due to its highly enriched PUFA content.     

Adhesion defective BHK cell mutant has cell surface heparan sulfate proteoglycan of altered properties

In the light of accumulating data that implicate cell surface heparan sulfate proteoglycans (HSPGs) with a role in cell interactions with extracellular matrix molecules such as fibronectin, we have compared the properties of these molecules in wild-type BHK cells and an adhesion-defective ricin-resistant mutant (RicR14). Our results showed that the mutant, unlike BHK cells, cannot form focal adhesions when adherent to planar substrates in the presence of serum. Furthermore, while both cell lines possess similar amounts of cell surface HSPG with hydrophobic properties, that of RicR14 cells had decreased sulfation, reduced affinity for fibronectin and decreased half-life on the cell surface when compared to the normal counterpart. Our conclusions based on this data are that these altered properties may, in part, account for the adhesion defect in the ricin-resistant mutant. Whether this results from the known alteration in assembly of N-linked glycans affecting the carbohydrate chains on the proteoglycan or some other combination of factors is discussed.     

OxLDL stimulates cell proliferation through a general induction of cell cycle proteins

Oxidized low-density lipoprotein (oxLDL) may be involved in atherosclerosis by stimulating proliferation of cells in the vessel wall. The purpose of this study was to identify the mechanism by which oxLDL induces proliferation. Quiescent human fibroblasts and rabbit smooth muscle cells were treated with 0, 10, or 50 microg/ml oxLDL for 24-48 h. This resulted in significant increases in total cell counts at both concentrations of oxLDL, at both time points, for both types of cells. Western blot analysis revealed that oxLDL-stimulated cell proliferation was associated with significant increases in the expression of proteins that regulate entry into and progression through the cell cycle [cell division cycle 2, cyclin-dependent kinase (cdk) 2, cdk 4, cyclin B1, cyclin D1, and PCNA]. Surprisingly, the expression of cell cycle inhibitors (p21 and p27) was stimulated by oxLDL as well, but this was to a lesser extent than the effects on cell cycle-activating proteins. OxLDL also induced nuclear localization of all cell cycle proteins examined. The similar effects of oxLDL on the translocation and expression of both cell cycle-activating and -inhibiting proteins may explain the controlled proliferative phenomenon observed in atherosclerosis as opposed to the more rapid proliferative event characteristic of cancer.     

Increase in nuclear calcium in smooth muscle cells exposed to oxidized low density lipoprotein

Vascular smooth muscle cells respond with an increase in intracellular Ca²⁺ within seconds after exposure to oxidized low density lipoprotein (oxLDL). This has been suggested to represent a signaling response that may have implications for gene expression. If so, oxLDL may induce increases in nuclear Ca²⁺ in smooth muscle cells in response to oxLDL. Aortic smooth muscle cells were exposed to 100 μg/ml oxLDL. Large, rapid increases in [Ca²⁺]_i were observed using fluo-3 as an indicator dye to detect intracellular Ca²⁺ on the stage of a confocal micro-scope. This was also confirmed using ratiometric imaging of indo signals. These elevations appeared to be localized to the nuclear region of the cell. DNA staining of the cells confirmed its localization to the nuclear / perinuclear region of the cell. Our data demonstrate that oxLDL induces a nuclear localized elevation in Ca²⁺i that may have important implications for nuclear function.     

Increase in nuclear calcium in smooth muscle cells exposed to oxidized low density lipoprotein

Vascular smooth muscle cells respond with an increase in intracellular Ca²⁺ within seconds after exposure to oxidized low density lipoprotein (oxLDL). This has been suggested to represent a signaling response that may have implications for gene expression. If so, oxLDL may induce increases in nuclear Ca²⁺ in smooth muscle cells in response to oxLDL. Aortic smooth muscle cells were exposed to 100 μg/ml oxLDL. Large, rapid increases in [Ca²⁺]_i were observed using fluo-3 as an indicator dye to detect intracellular Ca²⁺ on the stage of a confocal micro-scope. This was also confirmed using ratiometric imaging of indo signals. These elevations appeared to be localized to the nuclear region of the cell. DNA staining of the cells confirmed its localization to the nuclear / perinuclear region of the cell. Our data demonstrate that oxLDL induces a nuclear localized elevation in Ca²⁺i that may have important implications for nuclear function.