Safety, tolerability and toxicokinetics of the novel mitochondrial drug SUL-138 administered orally to rat and minipig.

Noncommunicable Chronic Diseases (NCD) are a socioeconomic burden and considered one of the major health challenges for coming decades. Mitochondrial dysfunction has been implicated mechanistically in their pathophysiology. Therefore, targeting mitochondria holds great promise to improve clinical outcomes in NCDs. SUL-138, an orally bioavailable small molecule efficacious from 0.5 mg/kg, improves mitochondrial function during disease in several preclinical animal models. As preparation for a First-in-Human (FIH) trial, SUL-138 was investigated in 30-day GLP repeated dose toxicity studies in rat and minipig, selected based on their comparability with human metabolism, to determine toxicokinetics, potential toxicity and its reversibility. Rats were allocated to either vehicle, 27, 136 or 682 mg/kg SUL-138 dose groups and minipigs were allocated to either vehicle, 16, 82 or 409 mg/kg. Treatment occurred orally for 30 days followed by a recovery period of 14 days. During these studies clinical observations, toxicokinetic, clinical pathology, necropsy and histopathology evaluations were performed. There was significant systemic exposure to SUL-138 and toxicokinetics was characterized by a rapid absorption and elimination. In the rat, toxicokinetics was dose-proportional and AUC0-tlast ratios in both species indicated that SUL-138 does not accumulate in vivo. No treatment-related adverse effects were observed for dose levels up to 136 and 82 mg/kg/day in rat and minipig respectively. In conclusion, these preclinical studies demonstrate that SUL-138 is well tolerated after repeated administration in rat and minipig, with NOAELs of 136 and 82 mg/kg/day, respectively.

Peri-Operative Kinetics of Plasma Mitochondrial DNA Levels during Living Donor Kidney Transplantation.

During ischemia and reperfusion injury (IRI), mitochondria may release mitochondrial DNA (mtDNA). mtDNA can serve as a propagator of further injury but in specific settings has anti-inflammatory capacities as well. Therefore, the aim of this study was to study the perioperative dynamics of plasma mtDNA during living donor kidney transplantation (LDKT) and its potential as a marker of graft outcome. Fifty-six donor-recipient couples from the Volatile Anesthetic Protection of Renal Transplants-1 (VAPOR-1) trial were included. Systemic venous, systemic arterial, and renal venous samples were taken at multiple timepoints during and after LDKT. Levels of mtDNA genes changed over time and between vascular compartments. Several donor, recipient, and transplantation-related variables significantly explained the course of mtDNA genes over time. mtDNA genes predicted 1-month and 24-month estimated glomerular filtration rate (eGFR) and acute rejection episodes in the two-year follow-up period. To conclude, mtDNA is released in plasma during the process of LDKT, either from the kidney or from the whole body in response to transplantation. While circulating mtDNA levels positively and negatively predict post-transplantation outcomes, the exact mechanisms and difference between mtDNA genes are not yet understood and need further exploration.

Hydrogen sulphide-induced hypometabolism in human-sized porcine kidneys.

BACKGROUND: Since the start of organ transplantation, hypothermia-forced hypometabolism has been the cornerstone in organ preservation. Cold preservation showed to protect against ischemia, although post-transplant injury still occurs and further improvement in preservation techniques is needed. We hypothesize that hydrogen sulphide can be used as such a new preservation method, by inducing a reversible hypometabolic state in human sized kidneys during normothermic machine perfusion. METHODS: Porcine kidneys were connected to an ex-vivo isolated, oxygen supplemented, normothermic blood perfusion set-up. Experimental kidneys (n = 5) received a 85mg NaHS infusion of 100 ppm and were compared to controls (n = 5). As a reflection of the cellular metabolism, oxygen consumption, mitochondrial activity and tissue ATP levels were measured. Kidney function was assessed by creatinine clearance and fractional excretion of sodium. To rule out potential structural and functional deterioration, kidneys were studied for biochemical markers and histology. RESULTS: Hydrogen sulphide strongly decreased oxygen consumption by 61%, which was associated with a marked decrease in mitochondrial activity/function, without directly affecting ATP levels. Renal biological markers, renal function and histology did not change after hydrogen sulphide treatment. CONCLUSION: In conclusion, we showed that hydrogen sulphide can induce a controllable hypometabolic state in a human sized organ, without damaging the organ itself and could thereby be a promising therapeutic alternative for cold preservation under normothermic conditions in renal transplantation.

Modulation of glutathione peroxidase activity by age-dependent carbonylation in glomeruli of diabetic mice.

AIMS: Low levels of reactive oxygen species and resulting oxidative protein modifications may play a beneficial role in cellular function under stress conditions. Here we studied the influence of age-dependent protein carbonylation on expression and activity of the anti-oxidative selenoenzyme glutathione peroxidase (GPx) in insulin-deficient Ins2Akita mice and type 2 diabetic obese db/db mice in context of diabetic nephropathy. METHODS: Protein carbonylation, GPx expression and activity were examined in kidney tissue and lysates by common histological and protein biochemical methods. RESULTS: In kidneys of Ins2Akita mice, carbonylated proteins, GPx-1 and GPx-4 expression were mainly detected in podocytes and mesangial cells. GPx activity was increased in kidney cortex homogenates of these mice. Remarkably, young animals did not show a concomitant increase in GPx expression but enhanced GPx carbonylation. No carbonylation-dependent modification of GPx activity was detected in db/db mice. In cultured podocytes hyperglycemia induced an increase in GPx expression but had no effect on activity or carbonylation. In kidney tissue sections of type 1 or type 2 diabetes patients, GPx-1 and GPx-4 expression but not overall protein carbonylation was significantly decreased. CONCLUSIONS: These results indicate the existence of a threshold for beneficial carbonylation-dependent redox signaling during the progression of diabetic nephropathy.

Renal endothelial function is associated with the anti-proteinuric effect of ACE inhibition in 5/6 nephrectomized rats.

In healthy rats, the physiological variation of baseline endothelial function of intrarenal arteries correlates with the severity of renal damage in response to a subsequent specific renal injury. However, whether such a variation in endothelial function may also condition or predict the variable response to angiotensin-converting enzyme-inhibiting treatment in these individuals has not been addressed before. To study this, 5/6 nephrectomy was performed to induce renal injury and chronic kidney disease in a group of healthy Wistar rats. At the time of nephrectomy, interlobar arteries were obtained from the extirpated right kidney and studied in vitro for endothelium-dependent relaxation to acetylcholine. Six weeks thereafter, treatment with lisinopril was started (n = 11) and continued for 9 wk. Proteinuria (metabolic cages) and systolic blood pressure (SBP; tail cuff) were evaluated weekly, and these were analyzed in relation to renal endothelial function at baseline. 5/6 Nephrectomy induced an increase in SBP and progressive proteinuria. Treatment with lisinopril reduced SBP and slowed proteinuria, albeit to a variable degree among individuals. The acetylcholine-induced renal artery dilation at baseline negatively correlated with lisinopril-induced reduction of proteinuria (r(2) = 0.648, P = 0.003) and with the decrease in SBP (r(2) = 0.592, P = 0.006). Our data suggest that angiotensin-converting enzyme-inhibitor attenuates the progression of renal damage the most in those individuals with decreased basal renal endothelial-mediated vasodilation.

Early Posttransplant Tryptophan Metabolism Predicts Long-term Outcome of Human Kidney Transplantation.

BACKGROUND: Chronic transplant dysfunction (CTD) is the leading cause of long-term loss of the renal allograft. So far, no single test is available to reliably predict the risk for CTD. Monitoring of tryptophan (trp) metabolism through indoleamine 2.3-dioxygenase (IDO) has been previously proposed to predict acute rejection of human kidney transplants. Here, we investigate the potential of IDO/trp degradation along the kynurenine (kyn) pathway to predict the long-term outcome of human kidney transplantation. METHODS: During the 2-year follow-up blood, urine, and kidney biopsies were collected from 48 renal transplant patients. Concentrations of kyn and trp in serum and urine were measured at 2 weeks, 6 months, and 2 years after transplantation. Kynurenine to tryptophan ratio was calculated as an estimate of trp degradation. To evaluate the histological changes and IDO expression, respectively, periodic acid schiff staining and immunohistochemistry for IDO were performed on biopsies taken at 6 months and 2 years. RESULTS: Two years after transplantation, kyn/trp was increased in urine and decreased in serum as compared to 2-week values. In 2-year biopsies, IDO expression was mainly found in infiltrating inflammatory cells and in the glomeruli. The urine level of trp 2 weeks after transplantation predicted the serum creatinine 6 months and the estimated creatinine clearance 2 years after transplantation. Additionally, serum level of kyn 6 months after transplantation predicted the serum creatinine 2 years after transplantation. CONCLUSIONS: Early serum and urine levels of trp and kyn may offer a novel route for early detection of patients at risk for developing CTD.

The role of endogenous H2S formation in reversible remodeling of lung tissue during hibernation in the Syrian hamster.

During hibernation, small mammals alternate between periods of metabolic suppression and low body temperature ('torpor') and periods of full metabolic recovery with euthermic temperatures ('arousal'). Previously, we demonstrated marked structural remodeling of the lung during torpor, which is rapidly reversed during arousal. We also found that cooling of hamster cells increased endogenous production of H(2)S through the enzyme cystathionine-ß-synthase (CBS). H(2)S suppresses the immune response and increases deposition of collagen. Therefore, we examined inflammatory markers and matrix metalloproteinase (MMP) activity in relation to CBS expression and H(2)S levels in lungs of euthermic and hibernating Syrian hamsters. Lung remodeling during torpor was confirmed by a strong increase in both collagenous and non-collagenous hydroxyproline content. The number of leukocytes in lung was unchanged in any phase of hibernation, while adhesion molecules VCAM-1 and ICAM-1, and the inflammatory marker NF-κB (P65) were modestly upregulated in torpor. Gelatinase activity was decreased in lungs from torpid animals, indicating inhibition of the Zn(2+)-dependent MMP-2 and MMP-9. Moreover, expression of CBS and tissue levels of H(2)S were increased in torpor. All changes normalized during arousal. Inhibition of gelatinase activity in torpor is likely caused by quenching of Zn(2+) by the sulphide ion of H(2)S. In accord, inhibition of CBS normalized gelatinase activity in torpid animals. Conversely, NaHS decreased the gelatinase activity of euthermic animals, which was attenuated by excess Zn(2+). Similar results were obtained on the activity of the Zn(2+)-dependent angiotensin converting enzyme. Our data indicate that increased production of H(2)S through CBS in hamster lungs during torpor contributes to remodeling by inhibition of gelatinase activity and possibly by suppression of the inflammatory response. Although administration of H(2)S is known to induce metabolic suppression in non-hibernating mammals ('suspended animation'), this is the first report implying endogenous H(2)S production in natural hibernation.

Carnosine prevents apoptosis of glomerular cells and podocyte loss in STZ diabetic rats.

BACKGROUND/AIMS: We identified carnosinase-1 (CN-1) as risk-factor for diabetic nephropathy (DN). Carnosine, the substrate for CN-1, supposedly is a protective factor regarding diabetic complications. In this study, we hypothesized that carnosine administration to diabetic rats might protect the kidneys from glomerular apoptosis and podocyte loss. METHODS: We examined the effect of oral L-carnosine administration (1g/kg BW per day) on apoptosis, podocyte loss, oxidative stress, AGEs and hexosamine pathway in kidneys of streptozotocin-induced diabetic Wistar rats after 3 months of diabetes and treatment. RESULTS: Hyperglycemia significantly reduced endogenous kidney carnosine levels. In parallel, podocyte numbers significantly decreased (-21% compared to non-diabetics, p<0.05), apoptotic glomerular cells numbers increased (32%, compared to non-diabetic, p<0.05) and protein levels of bax and cytochrome c increased (175% and 117%). Carnosine treatment restored carnosine kidney levels, prevented podocytes loss (+23% compared to diabetic, p<0.05), restrained glomerular apoptosis (-34% compared to diabetic; p<0.05) and reduced expression of bax and cytochrome c (-63% and -54% compared to diabetics, both p<0.05). In kidneys of all diabetic animals, levels of ROS, AGEs and GlcNAc-modified proteins were increased. CONCLUSION: By inhibition of pro-apoptotic signaling and independent of biochemical abnormalities, carnosine protects diabetic rat kidneys from apoptosis and podocyte loss.

Reversible remodeling of lung tissue during hibernation in the Syrian hamster.

During hibernation, small rodents such as hamsters cycle through phases of strongly suppressed metabolism with low body temperature (torpor) and full restoration of metabolism and body temperature (arousal). Remarkably, the repetitive stress of cooling-rewarming and hypoxia does not cause irreversible organ damage. To identify adaptive mechanisms protecting the lungs, we assessed histological changes as well as the expression and localization of proteins involved in tissue remodeling in lungs from Syrian hamsters at different phases of hibernation using immunohistochemical staining and western blot analysis. In torpor (early and late) phase, a reversible increased expression of smooth muscle actin, collagen, angiotensin converting enzyme and transforming growth factor-ß was found, whereas expression of the epidermal growth factor receptor and caveolin-1 was low. Importantly, all these alterations were restored during arousal. This study demonstrates substantial alterations in protein expression mainly in epithelial cells of lungs from hibernating Syrian hamsters. These structural changes of the bronchial airway structure are termed airway remodeling and often occur in obstructive lung diseases such as asthma, chronic obstructive pulmonary disease (COPD) and lung fibrosis. Unraveling the molecular mechanism leading to reversal of airway remodeling by the end of torpor may identify possible therapeutic targets to reduce progression of this process in patients suffering from asthma, chronic obstructive pulmonary disease and lung fibrosis.

Statement on national worklife priorities.

The National Institute for Occupational Safety and Health (NIOSH) WorkLife Initiative (WLI) [http://www.cdc.gov/niosh/worklife] seeks to promote workplace programs, policies, and practices that result in healthier, more productive employees through a focus simultaneously on disease prevention, health promotion, and accommodations to age, family, and life stage. The Initiative incorporates the Institute's foundational commitment to workplaces free of recognized hazards into broader consideration of the factors that affect worker health and wellbeing. Workplace hazards, such as physical demands, chemical exposures, and work organization, often interact with non-work factors such as family demands and health behaviors to increase health and safety risks. New workplace interventions being tested by the first three NIOSH WLI Centers of WorkLife Excellence are exploring innovative models for employee health programs to reduce the human, social, and economic costs of compromised health and quality of life. Many parties in industry, labor, and government share the goals of improving employee health while controlling health care costs. NIOSH convened a workshop in 2008 with representatives of the three Centers of Excellence to develop a comprehensive, long-range strategy for advancing the WorkLife Initiative. The recommendations below fall into three areas: practice, research, and policy. Responding to these recommendations would permit the WorkLife Center system to establish a new infrastructure for workplace prevention programs by compiling and disseminating the innovative practices being developed and tested at the Centers, and elsewhere. The WLI would also extend the customary scope of NIOSH by engaging with multiple NIH Institutes that are already generating research-to-practice programs involving the working-age population, in areas such as chronic disease prevention and management. Research to Practice (r2p) is a concept focused on the translation of research findings, technologies, and information into evidence-based prevention practices and products that are adopted in the workplace or other "real-world" settings. NIOSH's goal is to overcome the translational issues that now prevent state-of-the-art occupational health, health promotion, and chronic disease research findings from benefiting working age populations immediately, regardless of workplace size, work sector, or region of the country.

Endothelial cell ephrinB2-dependent activation of monocytes in arteriosclerosis.

OBJECTIVE: The expression of ephrinB2 in endothelial cells delineates their arterial phenotype and is a prerequisite for the development of the embryonic vasculature. Whereas the role of ephrinB2 in the microcirculation has been studied extensively, its expression and function in adult arteries is hardly understood. METHODS AND RESULTS: Our analyses showed that in mouse arteries, ephrinB2 is located on the luminal surface of endothelial cells and may physically interact with monocyte EphB receptors. Moreover, transdifferentiation of human monocytes into macrophages was associated with an increase in EphB2 expression, and exposing monocytes to immobilized ephrinB2 resulted in phosphorylation of the receptor followed by an increased expression of proinflammatory chemokines such as interleukin-8 and monocyte chemotactic protein-1/CCL2. Relating to the (patho)physiological relevance of these findings, immunofluorescence analyses revealed that ephrinB2 is most abundantly expressed in endothelial cells at arteriosclerosis predilection sites of the mouse aorta. Subsequent analyses indicated that monocyte adhesion to aortic segments abundantly expressing ephrinB2 is strongly enhanced and that endothelial cell ephrinB2 forward signaling is sufficient to upregulate cytokine expression in monocytes. CONCLUSIONS: These observations suggest a hitherto unknown link between vascular ephrinB2 expression and the proinflammatory activation of monocytes that may contribute to the pathogenesis of arteriosclerosis.

Heparin binding epidermal growth factor in renal ischaemia/reperfusion injury.

The epidermal growth factor (EGF) receptor and its ligands are crucially involved in the renal response to ischaemia. We studied the heparin binding-epidermal growth factor (HB-EGF), a major ligand for the EGF receptor, in experimental and human ischaemia/reperfusion injury (IRI). HB-EGF mRNA and protein expression was studied in rat kidneys and cultured human tubular (HK-2) cells that were subjected to IRI and in human donor kidneys during transplantation. The effect of EGF receptor inhibition was investigated in vivo and in vitro. Furthermore, urinary HB-EGF protein excretion was studied after renal transplantation. Finally, HB-EGF KO and WT mice were subjected to IRI to study the role of HB-EGF in renal injury. HB-EGF mRNA was significantly up-regulated in the early phase of IRI in rats, cells, and human donor biopsies. Treatment with PKI-166 reduces macrophage accumulation and interstitial alpha-SMA in the early phase of IRI in rats. In vitro, PKI-166 causes a marked reduction in HB-EGF-induced cellular proliferation. Urinary HB-EGF is increased after transplantation compared with control urines from healthy subjects. HB-EGF KO mice subjected to IRI revealed significantly less morphological damage after IRI, compared with WT mice. We conclude that IRI results in early induction of HB-EGF mRNA and protein in vivo and in vitro. Absence of HB-EGF and inhibition of the EGF receptor in the early phase of IRI has protective effects, suggesting a modulating role for HB-EGF.

Caveolin-1 is enriched in the peroxisomal membrane of rat hepatocytes.

UNLABELLED: Caveolae are a subtype of cholesterol-enriched lipid microdomains/rafts that are routinely detected as vesicles pinching off from the plasma membrane. Caveolin-1 is an essential component of caveolae. Hepatic caveolin-1 plays an important role in liver regeneration and lipid metabolism. Expression of caveolin-1 in hepatocytes is relatively low, and it has been suggested to also reside at other subcellular locations than the plasma membrane. Recently, we found that the peroxisomal membrane contains lipid microdomains. Like caveolin-1, hepatic peroxisomes are involved in lipid metabolism. Here, we analyzed the subcellular location of caveolin-1 in rat hepatocytes. The subcellular location of rat hepatocyte caveolin-1 was analyzed by cell fractionation procedures, immunofluorescence, and immuno-electron microscopy. Green fluorescent protein (GFP)-tagged caveolin-1 was expressed in rat hepatocytes. Lipid rafts were characterized after Triton X-100 or Lubrol WX extraction of purified peroxisomes. Fenofibric acid-dependent regulation of caveolin-1 was analyzed. Peroxisome biogenesis was studied in rat hepatocytes after RNA interference-mediated silencing of caveolin-1 and caveolin-1 knockout mice. Cell fractionation and microscopic analyses reveal that caveolin-1 colocalizes with peroxisomal marker proteins (catalase, the 70 kDa peroxisomal membrane protein PMP70, the adrenoleukodystrophy protein ALDP, Pex14p, and the bile acid-coenzyme A:amino acid N-acyltransferase BAAT) in rat hepatocytes. Artificially expressed GFP-caveolin-1 accumulated in catalase-positive organelles. Peroxisomal caveolin-1 is associated with detergent-resistant microdomains. Caveolin-1 expression is strongly repressed by the peroxisome proliferator-activated receptor-alpha agonist fenofibric acid. Targeting of peroxisomal matrix proteins and peroxisome number and shape were not altered in rat hepatocytes with 70%-80% reduced caveolin-1 levels and in livers of caveolin-1 knockout mice. CONCLUSION: Caveolin-1 is enriched in peroxisomes of hepatocytes. Caveolin-1 is not required for peroxisome biogenesis, but this unique subcellular location may determine its important role in hepatocyte proliferation and lipid metabolism.

Vascular contraction and preeclampsia: downregulation of the Angiotensin receptor 1 by hemopexin in vitro.

During normal pregnancy, in contrast to preeclampsia, plasma hemopexin activity is increased together with a decreased vascular angiotensin II receptor (AT(1)) expression. We now tested the hypothesis that hemopexin can downregulate the AT(1) receptor in vitro. Analysis of human monocytes or endothelial cells by flow cytometry showed decreased membrane density of AT(1) exclusively after incubation with active hemopexin, whereas in supernatants of these cell cultures, AT(1) molecules could be detected (dot blotting). Also, diminished AT(1) was observed in endothelial cell lysates after contact with hemopexin (Western blotting). Hemopexin also induced extracellular signal-regulated kinase 1/2 pathway inhibition in cells after stimulation with angiotensin II in vitro, indicating downregulation of AT(1) by hemopexin. In addition, functional loss of AT(1) occurred after incubation of rat aortic rings with active hemopexin, as reflected by decreased contraction of the aortic rings on stimulation with angiotensin II. It was further demonstrated that plasma from normal pregnant women decreased the AT(1) receptor expression on monocytes as compared with plasma from nonpregnant women or preeclamptic women. Finally, it was shown that plasma hemopexin activity increases during normal gestation from week 10 onward. We concluded that active hemopexin is able to downregulate the AT(1) receptor in human monocytes, endothelial cells, and rat aortic rings. We propose that the physiological role of enhanced hemopexin activity during healthy pregnancy is to downregulate the vascular AT(1) receptor, promoting an expanded vascular bed. Inhibition of hemopexin activity during preeclampsia may result in an enhanced AT(1) receptor expression and a contracted vascular bed.

Effects of ivabradine and metoprolol on cardiac angiogenesis and endothelial dysfunction in rats with heart failure.

Myocardial infarction (MI)-induced remodeling is associated with disturbed myocardial perfusion through vascular changes, such as reduced capillary density and endothelial dysfunction. Heart rate reduction (HRR) initiated immediately after MI stimulates angiogenesis and attenuates left ventricular dysfunction. We aimed to investigate the effects of long-term HRR on cardiac angiogenesis and endothelial function in a rat model of post-MI heart failure. Rats received early or late ivabradine or metoprolol for 12 or 9 weeks, respectively, and compared with untreated MI and sham animals 12 weeks after MI. Heart rate was measured in the conscious rat. MI resulted in an increased heart weight to body weight ratio, a decline in capillary density and a marked reduction in acetylcholine-induced relaxation. Early and late HRR by either ivabradine or metoprolol significantly increased capillary to myocyte ratio. Moreover, this ratio was significantly correlated to heart rate (r = -0.324 and P = 0.036). Neither early nor late chronic HRR prevented endothelial dysfunction, except a moderate improvement in late MI ivabradine group. In MI rats, HRR either by ivabradine or metoprolol treatment increases cardiac angiogenesis. Late HRR strategy was comparable to early HRR, suggesting that the beneficial effects are independent of the time of onset of therapy after MI.

Enhanced myogenic constriction of mesenteric artery in heart failure relates to decreased smooth muscle cell caveolae numbers and altered AT1- and epidermal growth factor-receptor function.

AIMS: We previously showed that enhanced myogenic constriction (MC) of peripheral resistance arteries involves active AT(1) receptors in chronic heart failure (CHF). Recent data suggest both transactivation of EGF receptors and caveolae-like microdomains to be implicated in the activity of AT(1) receptors. Thus, we assessed their roles in increased MC in mesenteric arteries of CHF rats. METHODS AND RESULTS: Male Wistar rats underwent myocardial infarction to induce CHF and were sacrificed after 12 weeks. The number of caveolae in smooth muscle cells (SMC) of mesenteric arteries of CHF rats was decreased by 43.6 +/- 4.0%, this was accompanied by increased MC, which was fully normalized to the level of sham by antagonists of the AT(1)-receptor (losartan) or EGF-receptor (AG1478). Acute disruption of caveolae in sham rats affected caveolae numbers and MC to a similar extent as CHF, however MC was only reversed by the antagonist of the EGF-receptor, but not by the AT(1)-receptor antagonist. Further, in sham rats, MC was increased by a sub-threshold concentration of angiotensin II and reversed by both AT(1)- as well as EGF-receptor inhibition. In contrast, increased MC by a sub-threshold concentration of EGF was only reversed by EGF receptor inhibition. CONCLUSION: These findings provide the first evidence that decreased SMC caveolae numbers are involved in enhanced MC in small mesenteric arteries, by affecting AT(1)- and EGF-receptor function. This suggests a novel mechanism involved in increased peripheral resistance in CHF.

Ultrasound and microbubble-targeted delivery of macromolecules is regulated by induction of endocytosis and pore formation.

Contrast microbubbles in combination with ultrasound (US) are promising vehicles for local drug and gene delivery. However, the exact mechanisms behind intracellular delivery of therapeutic compounds remain to be resolved. We hypothesized that endocytosis and pore formation are involved during US and microbubble targeted delivery (UMTD) of therapeutic compounds. Therefore, primary endothelial cells were subjected to UMTD of fluorescent dextrans (4.4 to 500 kDa) using 1 MHz pulsed US with 0.22-MPa peak-negative pressure, during 30 seconds. Fluorescence microscopy showed homogeneous distribution of 4.4- and 70-kDa dextrans through the cytosol, and localization of 155- and 500-kDa dextrans in distinct vesicles after UMTD. After ATP depletion, reduced uptake of 4.4-kDa dextran and no uptake of 500-kDa dextran was observed after UMTD. Independently inhibiting clathrin- and caveolae-mediated endocytosis, as well as macropinocytosis significantly decreased intracellular delivery of 4.4- to 500-kDa dextrans. Furthermore, 3D fluorescence microscopy demonstrated dextran vesicles (500 kDa) to colocalize with caveolin-1 and especially clathrin. Finally, after UMTD of dextran (500 kDa) into rat femoral artery endothelium in vivo, dextran molecules were again localized in vesicles that partially colocalized with caveolin-1 and clathrin. Together, these data indicated uptake of molecules via endocytosis after UMTD. In addition to triggering endocytosis, UMTD also evoked transient pore formation, as demonstrated by the influx of calcium ions and cellular release of preloaded dextrans after US and microbubble exposure. In conclusion, these data demonstrate that endocytosis is a key mechanism in UMTD besides transient pore formation, with the contribution of endocytosis being dependent on molecular size.

Caveolae and endothelial dysfunction: filling the caves in cardiovascular disease.

Discovery in the early 1990s of caveolin-1, the structural protein responsible for maintaining the ohm shape of caveolae, greatly enhanced investigations to elucidate the role of these little caves in the plasma membrane. Perhaps one of the most important realizations concerning caveolae and caveolin is that these elements play an important functional role in the modulation of cell signal transduction pathways, including those involved in endothelial nitric oxide synthase (eNOS) function. Their role was confirmed by studies with caveolin-1 knockout mice which lack caveolae and display abnormal endothelial function responses. One limitation of these knockout models, however, is that absence of the caveolin protein not only results in the lack of caveolae as a structure but also in the lack of interaction/modulation of enzymes/molecules (e.g. eNOS) to which caveolin binds (whether in- or outside caveolae). In contrast to caveolin knockout models, recent experimental findings suggest that in certain cardiovascular diseases caveolin may dissociate from caveolae to the cytosol, hence decreasing the number of caveolae without a change in the total amount of caveolin. Therefore, as the importance of defining the role of caveolins both in caveolae and in cellular regions is being highlighted, it seems also important at the same time to further define the role of caveolae per se being present in the plasma membrane as a structural entity. The objective of this review is to make an explorative tour on the role of caveolae in vascular endothelial function based on existing literature together with some preliminary experimental findings. Evidence and arguments are put forward that alterations in endothelial caveolae do occur in cardiovascular disease and may contribute to the observed endothelial dysfunction in these conditions.

Altered myogenic constriction and endothelium-derived hyperpolarizing factor-mediated relaxation in small mesenteric arteries of hypertensive subtotally nephrectomized rats.

OBJECTIVES: Chronic renal failure (CRF) is associated with altered systemic arterial tone and hypertension. Myogenic constriction and endothelium-derived hyperpolarizing factor (EDHF)-dependent relaxation represent major vasoregulatory mechanisms in small systemic arteries. Elevated myogenic response and impaired EDHF might participate in the development of essential hypertension; however, their role in CRF-related hypertension is unknown. We investigated whether myogenic response and EDHF are altered in subtotally nephrectomized (sNX) rats and whether these changes are modifiable by chronic treatment with angiotensin-converting enzyme (ACE) inhibitor. METHODS: In a pressure arteriograph, myogenic constriction and EDHF-mediated relaxation were evaluated in small mesenteric arteries isolated from male Wistar rats 15 weeks after either sham operation (n = 7) (SHAM), sNX (n = 12) or sNX followed by 9 weeks of treatment with lisinopril (sNX + LIS, 2.5 mg/kg, n = 13). RESULTS: Surprisingly, myogenic response was reduced in hypertensive CRF rats (maximal myogenic tone: 37 +/- 2 and 18 +/- 4%, P < 0.01; peak myogenic index: -0.80 +/- 0.08 and -0.40 +/- 0.12%/mmHg, P < 0.05 in SHAM and sNX respectively). At the same time EDHF-mediated relaxation was also impaired (maximal response: 92 +/- 2 and 77 +/- 5%, P < 0.01; pD2: 6.5 +/- 0.1 and 5.9 +/- 0.1, P < 0.05). Both myogenic response and EDHF were inversely related to the severity of renal failure and restored by treatment with lisinopril to levels found in SHAM animals. CONCLUSION: Major constrictive (myogenic) and dilatory (EDHF) mechanisms of small systemic arteries are impaired in hypertensive CRF rats. These alterations do not seem to participate in the development of hypertension, being rather directly related to the severity of renal impairment. Both systemic vascular changes might be restored by renoprotective treatment with ACE inhibitor.

Myocardial infarction enhances progressive renal damage in an experimental model for cardio-renal interaction.

Studied were the effects of myocardial infarction (MI) on mild renal function loss in unilateral nephrectomized (UnX) rats. UnX was performed, followed after 1 wk by a variable MI (UnX + MI; n = 24). Rats with only UnX (n = 15) or MI (n = 9) and double sham animals (CON, n = 15) served as controls. Renal outcome was measured by proteinuria and plasma creatinine. Focal glomerulosclerosis (FGS) incidence was evaluated by renal histology. Cardiac function and systolic BP were measured. A division into small and large infarcts after UnX was made a priori, resulting in two groups, one with a mild MI (<20%; n = 15) and one with a moderate MI (>20%; n = 9). Mild proteinuria up to 55.5 mg/d was observed in the UnX + mild MI group, whereas proteinuria rose significantly higher to 124.5 mg/d in the UnX + moderate MI group. Incidence of FGS was significantly increased in both UnX + MI groups compared with all other groups. The average MI size was 18%, 17%, and 25% in the MI, UnX + mild MI, and UnX + moderate MI group, respectively. LVP in both UnX + MI groups was correlated with proteinuria, indicative of a cardio-renal interaction. Clinically, these data imply that more patients are at risk for cardiovascular events and that after such an event, their chance of more renal function loss increases. Finding the underlying mechanism will enable improved protection for both kidneys and heart.