Familial hypercholesterolemia (FH) is a condition caused by mutations in the low-density lipoprotein receptor (LDLR) gene. Expression of LDLR is highly regulated and excess receptor expression is cytotoxic. To incorporate essential gene regulation into a gene therapy vector for FH, we generated vectors in which the expression of therapeutic human LDLR gene, or luciferase reporter gene, is driven by 10 kb of human LDLR genomic DNA encompassing the promoter region including elements essential for physiologically regulated expression. Using luciferase expression and specific LDL binding and internalization assays, we have shown in vitro that the genomic promoter element confers long-term, physiologically regulated gene expression and complementation of receptor deficiency in culture for 240 cell-generations. This was demonstrated in the presence of sterols or statins, modifiers of LDLR promoter activity. In vivo, we demonstrate efficient liver-specific delivery and expression of luciferase following hydrodynamic tail-vein injection and confirm that expression from the LDLR promoter element is sensitive to statin administration. We also demonstrate long-term LDLR expression from the 10-kb promoter element up to 9 months following delivery. The vector system that we describe provides the efficient delivery, long-term expression, and physiological regulation required for a successful gene therapy intervention for FH.
Peroxisome proliferation in the liver is a well-documented response that occurs in some species upon treatment with hypolipidemic drugs, such as fibrates. Typically, liver peroxisome proliferation has been estimated by direct counting via electron microscopy, as well as by gene expression, enzyme activity, and immunolabeling. We have developed a novel method for the immunofluorescent labeling of peroxisomes, using an antibody to the 70-kDa peroxisomal membrane protein (PMP70) coupled with fluorescent nanocrystals, Quantum Dots. This method is applicable to standard formalin-fixed, paraffin-embedded tissues. Using this technique, a dose-dependent increase in PMP70 labeling was evident in formalin-fixed liver sections from fenofibrate-treated rats. In formalin-fixed liver sections from cynomolgus monkeys given ciprofibrate, quantitative image analysis showed a statistically significant increase in PMP70 labeling compared to control; the increase in hepatic PMP70 protein levels was corroborated by immunoblotting using total liver protein. An increase in hepatic peroxisome number in ciprofibrate-treated monkeys was confirmed by electron microscopy. An advantage of the Quantum Dot/PMP70 method is that a single common protocol can be used to label peroxisomes from several different species, and many of the common problems that arise with immunolabeling, such as fading and low signal strength, are eliminated.
Melanosomes (pigment granules) within retinal pigment epithelial (RPE) cells of fish and amphibians undergo massive migrations in response to light conditions to control light flux to the retina. Previous research has shown that melanosome motility within apical projections of dissociated fish RPE cells requires an intact actin cytoskeleton, but the mechanisms and motors involved in melanosome transport in RPE have not been identified. Two in vitro motility assays, the Nitella assay and the sliding filament assay, were used to characterize actin-dependent motor activity of RPE melanosomes. Melanosomes applied to dissected filets of the Characean alga, Nitella, moved along actin cables at a mean rate of 2 microm/min, similar to the rate of melanosome motility in dissociated, cultured RPE cells. Path lengths of motile melanosomes ranged from 9 to 37 microm. Melanosome motility in the sliding filament assay was much more variable, ranging from 0.4-33 microm/min; 70% of velocities ranged from 1-15 microm/min. Latex beads coated with skeletal muscle myosin II and added to Nitella filets moved in the same direction as RPE melanosomes, indicating that the motility is barbed-end directed. Immunoblotting using antibodies against myosin VIIa and rab27a revealed that both proteins are enriched on melanosome membranes, suggesting that they could play a role in melanosome transport within apical projections of fish RPE.
Perivascular adipose tissue (PVAT) exerts an anti-contractile effect which is vital in regulating blood pressure. Evidence suggests that the sympathetic nervous stimulation of PVAT triggers the release of anti-contractile factors via activation of beta 3 -adrenoceptors. There is considerable evidence of sympathetic over-activity in obesity, which could result in the loss of PVAT function, and subsequent hypertension. Therefore it was decided to examine beta 3 -adrenoceptor function in obesity.Electrical field stimulation (EFS) profiles of healthy and obese mouse mesenteric arteries (<200 um, +/-PVAT) were characterised using wire myography (0.1-30 Hz, 20V, 0.2 ms pulse duration, 4s train duration). To demonstrate the release of an anti-contractile factor in health, the solution surrounding stimulated exogenous PVAT was transferred to a PVAT denuded vessel. Beta 3-adrenoceptor function was investigated using the agonist CL-316,243 (10uM) and antagonist SR59203A (100nM). The role of the vasodilator nitric oxide (NO) was studied using nitric oxide synthase (NOS) inhibitor L-NMMA (100uM), and NOS activator histamine (100uM).During EFS healthy PVAT elicits an anti-contractile effect (n = 8, P < 0.001); however the anti-contractile function of obese PVAT is lost (n = 8, P = 0.35). Inhibition of beta 3-adrenoceptors in healthy PVAT using SR59230A significantly reduced the anti-contractile effect (n = 8, P < 0.01), whereas activation of beta 3-adrenoceptors in obese PVAT using CL-316,243 did not restore function (n = 7, P = 0.77). Solution transfer from stimulated healthy exogenous PVAT to a -PVAT vessel significantly reduced contraction (n = 8, P < 0.01), confirming that stimulated PVAT releases a transferable anticontractile factor. The release of this factor could be inhibited using SR59230A (n = 7, P = 0.47). Solution transfer from obese PVAT had no effect on contraction (n = 6, P = 0.41), and again could not be restored using CL-316,243 (n = 6, P = 0.14). In healthy PVAT, inhibition of NOS using L-NMMA abolished the anti-contractile effect (n = 8, P < 0.01). In obese PVAT, activation of NOS using histamine was able to restore the anti-contractile function (n = 4, P < 0.05).These results demonstrate that in health PVAT releases an anti-contractile factor via activation of beta 3 -adrenoreceptors, which downstream trigger the release of NO. In obesity, the anti-contractile effect is lost and cannot be restored by beta 3 -adrenoceptor activation, but is restored by activation of NOS. This suggests that in obesity beta 3 -adrenoreceptors must be downregulated or desensitised, leading to a loss of anti-contractile function, which may contribute to the development of hypertension.
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