OS067. VEGF, a novel stimulator of aldosterone production.

INTRODUCTION: Adrenal aldosterone production depends upon capillary integrity. Inadequately explained by increased renin secretion, aldosterone is high in pregnancy, a proangiogenic state. In preeclampsia, low aldosterone levels coincide with disturbed endothelial integrity due to disrupted VEGF signaling. OBJECTIVES: We hypothesized that the stimulation of adrenal aldosterone production is VEGF-sensitive. METHODS: We cultured endothelial cells (EC) in the presence and absence of VEGF. The supernatent was transferred to cultured adrenal cells, either the cell line H295R or isolated primary human adrenal cells from zona glomerulosa. aldosterone synthase mRNA and protein expression, aldosterone synthesis was assessed by adding radioactive labeled precursors or measuring aldosterone in the supernatent by Elisa. Cells were cultured either with angiotensin II (Ang II), VEGF or a combination hereof. Adenovirus-based overexpression of the soluble VEGF receptor type 1 (sFlt-1) was used to simulated conditions of preeclampsia in rats and its effect on the adrenocortical vasculature and circulating aldosterone levels. RESULTS: EC conditioning in the presence of VEGF enhanced aldosterone synthase activity in human adrenocortical cells. VEGF either alone or combined with Ang II increased aldosterone synthase transcription, enzyme availability and aldosterone production in adrenal cells. Neuropilin-1 and VEGF receptor expression differed only for Flt-1 which was present in ECs but not in adrenocortical cells. In contrast to Ang II, VEGF did not upregulate the steroidogenic acute regulatory protein. In line with this observation, Ang II stimulated both aldosterone and cortisol synthesis from progesterone whereas VEGF preferably the former. In rats, overexpression of sFlt-1 which traps VEGF led to adrenocortical capillary rarefaction. Serum aldosterone concentrations inversely correlated with sFlt-1 levels. CONCLUSION: In conclusion, VEGF stimulates aldosterone production indirectly via ECs and directlyin adrenocortical cells a finding explaining the increased aldosterone/renin ratio in normal pregnancy. It is reasonable to assume that the inappropriately low aldosterone availability in preeclampsia is a consequence of the known disturbed VEGF signaling.

Circulatory and renal consequences of pregnancy in diabetic NOD mice.

OBJECTIVES: Women with diabetes have elevated gestational risks for severe hemodynamic complications, including preeclampsia in mid- to late pregnancy. This study employed continuous, chronic radiotelemetry to compare the hemodynamic patterns in non-obese diabetic (NOD) mice who were overtly diabetic or normoglycemic throughout gestation. We hypothesized that overtly diabetic, pregnant NOD mice would develop gestational hypertension and provide understanding of mechanisms in progression of this pathology. STUDY DESIGN: Telemeter-implanted, age-matched NOD females with and without diabetes were assessed for six hemodynamic parameters (mean, systolic, diastolic, pulse pressures, heart rate and activity) prior to mating, over pregnancy and over a 72 h post-partum interval. Urinalysis, serum biochemistry and renal histopathology were also conducted. RESULTS: Pregnant, normoglycemic NOD mice had a hemodynamic profile similar to other inbred strains, despite insulitis. This pattern was characterized by an interval of pre-implantation stability, post implantation decline in arterial pressure to mid gestation, and then a rebound to pre-pregnancy baseline during later gestation. Overtly diabetic NOD mice had a blood pressure profile that was normal until mid-gestation then become mildly hypotensive (-7 mmHg, P < 0.05), severely bradycardic (-80 bpm, P < 0.01) and showed signs of acute kidney injury. Pups born to diabetic dams were viable but growth restricted, despite their mothers' failing health, which did not rebound post-partum (-10% pre-pregnancy pressure and HR, P < 0.05). CONCLUSIONS: Pregnancy accelerates circulatory and renal pathologies in overtly diabetic NOD mice and is characterized by depressed arterial pressure from mid-gestation and birth of growth-restricted offspring.

Effects of cigarette smoke on salivary protein tyrosine nitration.

INTRODUCTION: Nitration of tyrosine and tyrosine-containing proteins and their roles in pathophysiology have just recently been reviewed. Despite low yields of tyrosine modifications, nitration of tyrosine residues may inactivate important proteins. Nitrotyrosine can be formed by various nitrating agents, including peroxynitrite. Thus, the occurrence of nitrotyrosine-containing proteins in vivo should be regarded as a general indication of tissue damage induced by reactive nitrogen species such as peroxynitrite. This strongly suggests that peroxynitrite could be formed in vivo under certain pathophysiological conditions. OBJECTIVE: Our aim in this study was to elucidate the effect of cigarette smoke (CS) on nitrotyrosine formation in saliva proteins. METHODS: We exposed saliva to CS, in vitro, and used Western Blotting (WB) and monoclonal anti-nitrotyrosine antibody to assess the level of saliva protein nitration. RESULTS: As saliva contains extensive amounts of nitrites, it was no surprise that at basal levels, saliva proteins, albumin, and α-amylase all were already nitrated. The WB also revealed that with continuous exposure to CS the tyrosine nitration of both albumin and α-amylase is declining significantly after 3 h. A quite similar effect was seen after exposure to aldehydes, but to a less extent as compared to CS. Exposure of nitrotyrosine-modified bovine serum albumin (BSA-N) to aldehydes, produced a similar effect, meaning a decrease in tyrosine nitration. CONCLUSIONS: These findings might be explained by the possible ability of CS aldehydes to reduce protein-bound nitro group to an amine. Another proposed mechanism is that CS unsaturated aldehydes react with proteins mainly through Michael addition reaction; leading to the generation of stable aldehyde-protein adducts (APA). Thus, it may react with nitro groups of saliva proteins, like albumin or α-amylase, to generate APA, which ultimately, may not be recognized by our antibody. Another possible mechanism, is interaction between the aldehyde group with the hydroxyl group of the 3-nitrotyrosine, forming a hemiacetal, which is not recognized by the antibody. This mechanism might explain the difference in the denitration effects caused by the saturated aldehyde acetaldehyde, which exists in large amounts in CS, and unsaturated aldehydes. Therefore, it is possible that the main player in the CS smoke denitration effect on salivary proteins is the aldehyde group and not the double bond of unsaturated aldehydes.

Effects of cigarette smoke borne reactive nitrogen species on salivary alpha-amylase activity and protein modifications.

Cigarette smoke (CS) is associated with a variety of human pathologies including cardiovascular disease and cancer. Oral squamous cell carcinoma (OSCC) is the most common malignancy of the head and neck. The major inducer of OSCC is exposure to tobacco. Recent studies demonstrated that oxidative and nitrosative stress contributes to the development of oral carcinogenesis through DNA damage. All salivary reactive nitrogen species (RNS) analyzed from OSCC patients are significantly higher in comparison with healthy subjects. Our findings show that CS and external RNS addition induced reduction in alpha-amylase activity and produced some excited carbonyl formation, but to a much less extant than CS. The addition of epigallocatechine-3-gallate (EGCG) to saliva produced no protective effect against damage to alpha-amylase activity. Our proposed mechanism for the decrease in alpha-amylase activity is the formation of adducts at SH groups of the alpha-amylase active site. In this case, EGCG was unable to counteract this phenomenon, as it does not reduce the concentration of disulfides, and does not alter the amount of protein-SH moieties. However, EGCG did reduce the levels of excited carbonyl formation. Our results indicate that although RNS are abundant in CS, a significant decrease in amylase activity is due to other components in CS, probably aldehydes, reacting with the thiol group of proteins by the Michael addition reaction.

Inhibition of salivary amylase activity by cigarette smoke aldehydes.

Cigarette smoke (CS) is a leading known cause of cancer and cardiovascular diseases worldwide. The mechanisms by which CS produces its damaging effects seem to be multifactorial. Among others, CS toxicity is due also to several compounds like alpha,beta-unsaturated aldehydes (acrolein, crotonaldehyde) and saturated aldehydes (acetaldehyde). Aldehydes could interact with thiol compounds of salivary proteins, leading to structural and functional alterations of these molecules. Prior in vitro studies have shown that there is a significant decrease in several known enzymatic activities following exposure to CS. Additionally, it was found that glutathione (GSH) has protective effect against the damaging role of CS to salivary enzymes, emphasizing the role of thiol groups in the mechanism of inactivation of these enzymes. In this study, salivary amylase activity showed a significant inhibition following exposure to CS, and to external addition of purified aldehydes known to be present in CS, due probably to the interaction between aldehydes and -SH groups of the enzyme. Our results indicate that although saturated aldehydes are the chief aldehydes present in CS, a significant decrease in amylase activity was due to unsaturated aldehydes, reacting, probably, through their double bond with the thiol group of proteins by the Michael addition reaction.