SummaryTo determine if short-term calorie restriction reverses vascular endothelial dysfunction in old mice, old (O, n = 30) and young (Y, n = 10) male B6D2F1 mice were fed ad libitum (AL) or calorie restricted (CR, approximately 30%) for 8 weeks. Ex vivo carotid artery endothelium-dependent dilation (EDD) was impaired in old ad libitum (OAL) vs. young ad libitum (YAL) (74 ± 5 vs. 95 ± 2% of maximum dilation, P < 0.05), whereas old calorie-restricted (OCR) and YCR did not differ (96 ± 1 vs. 94 ± 3%). Impaired EDD in OAL was mediated by reduced nitric oxide (NO) bioavailability associated with decreased endothelial NO synthase expression (aorta) (P < 0.05), both of which were restored in OCR. Nitrotyrosine, a cellular marker of oxidant modification, was markedly elevated in OAL (P < 0.05), whereas OCR was similar to Y. Aortic superoxide production was 150% greater in OAL vs. YAL (P < 0.05), but normalized in OCR, and TEMPOL, a superoxide dismutase (SOD) mimetic that restored EDD in OAL (to 97 ± 2%), had no effect in Y or OCR. OAL had increased expression and activity of the oxidant enzyme, NADPH oxidase, and its inhibition (apocynin) improved EDD, whereas NADPH oxidase in OCR was similar to Y. Manganese SOD activity and sirtuin1 expression were reduced in OAL (P < 0.05), but restored to Y in OCR. Inflammatory cytokines were greater in OAL vs. YAL (P < 0.05), but unaffected by CR. Carotid artery endothelium-independent dilation did not differ among groups. Short-term CR initiated in old age reverses age-associated vascular endothelial dysfunction by restoring NO bioavailability, reducing oxidative stress (via reduced NADPH oxidase-mediated superoxide production and stimulation of anti-oxidant enzyme activity), and upregulation of sirtuin-1.

A senescent phenotype in endothelial cells is associated with increased apoptosis, reduced endothelial nitric oxide synthase (eNOS) and inflammation, which are implicated in arterial dysfunction and disease in humans. We tested the hypothesis that changes in microRNAs are associated with a senescent phenotype in human aortic endothelial cells (HAEC). Compared with early-passage HAEC, late-passage HAEC had a reduced proliferation rate and increased staining for senescence-associated beta-galactosidase and the tumor suppressor p16INK4a. Late-passage senescent HAEC had reduced expression of proliferation-stimulating/apoptosis-suppressing miR-21, miR-214 and miR-92 and increased expression of tumor suppressors and apoptotic markers. eNOS-suppressing miR-221 and miR-222 were increased and eNOS protein and eNOS activation (phosphorylation at serine1177) were lower in senescent HAEC. Caveolin-1 inhibiting miR-133a was reduced and caveolin-1, a negative regulator of eNOS activity, was elevated in senescent HAEC. Inflammation-repressing miR-126 was reduced and inflammation–stimulating miR-125b was increased, whereas inflammatory proteins were greater in senescent HAEC. Development of a senescent arterial endothelial cell phenotype featuring reduced cell proliferation, enhanced apoptosis and inflammation and reduced eNOS is associated with changes in miRNAs linked to the regulation of these processes. Our results support the hypothesis that miRNAs could play a critical role in arterial endothelial cell senescence.

The purpose of the present study was to assess the role of diffusion and convection during filtration of Ficoll across the glomerular filter by comparing glomerular sieving coefficients (theta) to neutral fluorescein isothiocyanate (FITC)-Ficoll 70/400 obtained at low (hydropenic) vs raised (normal) glomerular filtration rates (GFRs). The theta for FITC-Ficoll was determined in anesthetized Wistar rats (304 +/- 18 g) following laparotomy and cannulation of the ureters, used for urine sampling. After surgery, GFR was 1.2 +/- 0.16 ml/min (+/- s.e.), assessed using the plasma to urine clearance of FITC-inulin and (51)Cr-ethylenediaminetetraacetic acid. FITC-Ficoll 70/400 was infused intravenously (i.v.) following an initial bolus dose. To raise GFR, to an average of approximately 2 ml/min, 5 ml of serum together with glucagon (3 microg/min) was given i.v. FITC-inulin and FITC-Ficoll were determined in plasma and urine using size-exclusion high-performance liquid chromatography. The theta for Ficoll as a function of Stokes-Einstein radius was significantly reduced in the range of 13-43 A when GFR was raised. The maximal theta lowering effect, in relative terms, of raising GFR was obtained for a Ficoll a(e) of approximately 32 A. For Ficoll(36 A) (cf. albumin), theta was reduced from 0.111+/- 0.009 to 0.081+/- 0.012 (P < 0.05; n = 7) for the GFR increment imposed. The reduction in theta for Ficoll after raising GFR indicates the presence of a high diffusive component of glomerular Ficoll filtration in rats in vivo and contradicts the notion of a significant concentration polarization effect in the glomerular filter upon Ficoll molecules < 50 A in radius.

This study was performed to evaluate the alterations of glomerular filtration barrier characteristics following acute renal ischemia-reperfusion (I/R). Ischemia was induced in anesthetized rats by unilateral renal artery occlusion for either 20 or 60 min, followed by reperfusion during 20 or 60 min, respectively, with the contralateral kidney serving as control. Sieving coefficients (theta) were obtained by analyzing Ficoll [mol.radius (a(e)) 13-85 A] in urine and plasma after 20 and 60 min I/R. Furthermore, theta for human serum albumin (HSA) was estimated using a tissue uptake technique after 20 and 60 min of I/R, while clearance of HSA compared with that for neutralized HSA (nHSA) was assessed after 20 min of I/R only. Glomerular filtration rate (GFR) was measured by [51Cr]EDTA and inulin. I/R reduced GFR and increased theta for Ficoll molecules of a(e)>55 A and theta for albumin. theta for Ficoll vs. a(e), analysed using a two-pore model, demonstrated that, despite increases in theta, the large-pore fractional ultrafiltration coefficient (alpha(L)) was unchanged after 20 min of I/R, owing to the decline in GFR, but increased after 60 min of I/R. However, the apparent alpha(L) for albumin increased already after 20 min of I/R (P < 0.005) and the nHSA/HSA clearance ratio was slightly reduced, possibly reflecting a diminished negative charge barrier. In conclusion, after 20 min of I/R, indications of a reduced charge selectivity were noted, while after 60 min of I/R, there was mainly a reduction in size selectivity, compatible with an increased formation of large pores.

Nonshivering thermogenesis induced in brown adipose tissue (BAT) during high-fat feeding is mediated through uncoupling protein 1 (UCP1). UCP2 is a recently identified homologue found in many tissues. To determine the role of UCP1 and UCP2 in thermoregulation and energy balance, we investigated the long-term effect of high-fat feeding on mRNA levels in mice at two different ambient temperatures. We also treated mice with the anorectic peptide enterostatin and compared mRNA levels in BAT, white adipose tissue (WAT), stomach, and duodenum. Here, we report that high-fat feeding at 23 degrees C increased UCP1 and UCP2 levels in BAT four- and threefold, respectively, and increased UCP2 levels fourfold in WAT. However, at 29 degrees C, UCP1 decreased, whereas UCP2 remained unchanged in BAT and increased twofold in WAT. Enterostatin increased UCP1 and decreased UCP2 mRNA in BAT. In stomach and duodenum, high-fat feeding decreased UCP2 mRNA, whereas enterostatin increased it. Our results suggest that the regulation of uncoupling protein mRNA levels by high-fat feeding is dependent on ambient temperature and that enterostatin is able to modulate it.

It is established that the glomerular filter sieves macromolecules based on their size, shape, and charge. Anionic proteins are thus retarded compared with their neutral or cationic counterparts. However, recent studies have indicated that charge effects are small, or even "anomalous," for polysaccharides. We therefore investigated the impact of charge on the glomerular permeability to polysaccharides by comparing sieving coefficients (theta; primary urine-to-plasma concentration ratio) for negatively charged, carboxymethylated (CM) FITC-Ficoll and FITC-dextran with their neutral counterparts. For these probes, theta were determined in anesthetized Wistar rats [269 +/- 2.7 g (+/-SE; n = 36)], whose ureters were cannulated for urine sampling. The glomerular filtration rate was assessed using FITC-inulin. Polysaccharides were constantly infused, and after equilibration, urine was collected and a midpoint plasma sample was taken. Size and concentration determinations of the FITC-labeled polysaccharides were achieved by size-exclusion HPLC (HPSEC). For CM-Ficoll, theta was significantly increased (32 times at 55 A) compared with that of uncharged Ficoll. A small increase in theta for CM-dextran compared with neutral dextran was also observed (1.8 times at 55 A). In conclusion, negatively charged Ficoll relative to neutral Ficoll was found to be markedly hyperpermeable across the glomerular filter. Furthermore, negatively charged Ficoll was observed to be larger on HPSEC compared with its neutral counterpart of the same molecular weight. It is proposed that the introduction of negative charges in the "dendrimeric," cross-linked Ficoll molecule may alter its configuration, so as to make it more extended, and conceivably, more flexible, thereby increasing its glomerular permeability.

Caveolae are membrane organelles that play roles in glucose and lipid metabolism and in vascular function. Formation of caveolae requires caveolins and cavins. The make-up of caveolae and their density is considered to reflect cell-specific transcriptional control mechanisms for caveolins and cavins, but knowledge regarding regulation of caveolae genes is incomplete. Myocardin (MYOCD) and its relative MRTF-A (MKL1) are transcriptional coactivators that control genes which promote smooth muscle differentiation. MRTF-A communicates changes in actin polymerization to nuclear gene transcription. Here we tested if myocardin family proteins control biogenesis of caveolae via activation of caveolin and cavin transcription. Using human coronary artery smooth muscle cells we found that jasplakinolide and latrunculin B (LatB), substances that promote and inhibit actin polymerization, increased and decreased protein levels of caveolins and cavins, respectively. The effect of LatB was associated with reduced mRNA levels for these genes and this was replicated by the MRTF inhibitor CCG-1423 which was non-additive with LatB. Overexpression of myocardin and MRTF-A caused 5-10-fold induction of caveolins whereas cavin-1 and cavin-2 were induced 2-3-fold. PACSIN2 also increased, establishing positive regulation of caveolae genes from three families. Full regulation of CAV1 was retained in its proximal promoter. Knock down of the serum response factor (SRF), which mediates many of the effects of myocardin, decreased cavin-1 but increased caveolin-1 and -2 mRNAs. Viral transduction of myocardin increased the density of caveolae 5-fold in vitro. A decrease of CAV1 was observed concomitant with a decrease of the smooth muscle marker calponin in aortic aneurysms from mice (C57Bl/6) infused with angiotensin II. Human expression data disclosed correlations of MYOCD with CAV1 in a majority of human tissues and in the heart, correlation with MKL2 (MRTF-B) was observed. The myocardin family of transcriptional coactivators therefore drives formation of caveolae and this effect is largely independent of SRF.

From these results it is concluded that for flexible and asymmetric macromolecules, their degree of glomerular hyperpermeability is proportional to their degree of 'molecular extension'. Thus, compared with globular proteins, the polysaccharides investigated, including Ficoll, were found to be hyperpermeable across the glomerular filter in vivo.