Branko Braam

Perinatal l-Arginine and Antioxidant Supplements Reduce Adult Blood Pressure in Spontaneously Hypertensive Rats

Embryo cross-transplantation and cross-fostering between spontaneously hypertensive rats (SHR) and normotensive rats (WKY) suggest that perinatal environment modulates the genetically determined phenotype. In SHR the balance between NO and reactive oxygen species (ROS) is disturbed. We hypothesized that increasing NO and diminishing ROS in perinatal life would ameliorate hypertension in adult SHR. Pregnant SHR and WKY and their offspring received L-arginine plus antioxidants (vitamin C, vitamin E, and taurine) during the last 2 weeks of pregnancy and then until either 4 or 8 weeks after birth. Systolic blood pressure (SBP) and urinary excretion of protein, nitrates (NOx), and thiobarbituric acid reactive substances (TBARS) were measured. At 48 weeks of age rats were euthanized for glomerular counts. Perinatal supplements reduced SBP persistently in SHR and prevented the SBP increase observed in aging WKY. Initially NOx excretion was lower and TBARS excretion higher in SHR than WKY. There was a direct effect on NOx excretion in supplemented pregnant SHR and their offspring, but no increase was observed after stopping the supplements. TBARS excretion was only depressed up to 14 weeks by the supplements despite persistent differences in SBP. Consistent effects on nephron number were absent. Mild proteinuria, present in control SHR at 48 weeks, was prevented in all supplemented rats. Perinatal supplementation of NO substrate and antioxidants results in persistent reduction of SBP and renal protection in SHR, although effects on NOx and TBARS were only transient. This suggests a critical role for perinatal pro- and antioxidant balance in programming BP later in life.

Fibronectin is a hypoxia-independent target of the tumor suppressor VHL

The von Hippel–Lindau (VHL) tumor suppressor gene regulates the extracellular matrix by controlling fibronectin deposition. To identify novel VHL target genes, we subjected mRNA from VHL‐deficient RCC cells (786‐0‐pRC) and a transfectant re‐expressing wildtype VHL (786‐0‐VHL) to differential expression profiling. Among the differentially expressed genes, we detected that fibronectin is upregulated in the presence of VHL, while it is not affected by hypoxia. Thus regulation of fibronectin deposition by VHL occurs at the transcriptional level, irrespective of oxygen levels.

Myofibroblast progenitor cells are increased in number in patients with type 1 diabetes and express less bone morphogenetic protein 6: a novel clue to adverse tissue remodelling?

Aims/hypothesisGrowth factor imbalance and endothelial progenitor cell dysfunction are well-known elements of the inappropriate response to injury in human and experimental diabetes. We hypothesised that in diabetes the outgrowth of myofibroblast progenitor cells (MFPCs) is also altered and that this relates to aberrant gene expression of growth factors involving members of the TGF-β/bone morphogenetic protein (BMP) superfamily.Subjects and methodsMFPCs were cultured from peripheral blood mononuclear cells of patients with type 1 diabetes and control subjects. Microarray analysis, quantitative PCR and ELISA were used to identify differentially regulated TGF-β/BMP superfamily genes in diabetes- and control-derived MFPC. Possible effects of BMP6 on TGF-β-induced gene expression were examined in cultured renal fibroblasts (TK173 cells).ResultsBlood from diabetic patients yielded higher numbers of MFPCs than blood from control subjects (1.6-fold increase, p<0.05), involving increased proliferation and decreased apoptosis. BMP6 mRNA and protein were downregulated in MFPCs derived from patients with diabetes (3.9- and 1.8-fold decrease, respectively, p<0.05). Furthermore, an inverse correlation was observed between BMP6 mRNA level and the number of MFPCs in patients with diabetes (r=−0.85, p<0.05). In TK173 cells, BMP6 antagonised the TGF-β-induced expression of the genes encoding plasminogen activator inhibitor-1 and connective tissue growth factor (70 and 50% reduction, respectively).Conclusions/interpretationConsidering the importance of BMP6 in processes such as angiogenesis and its novel anti-TGF-β effects, we propose that the excess numbers of BMP6-deficient MFPCs may favour adverse tissue remodelling in patients with diabetes, both numerically and by inappropriate orchestration of their microenvironment.

Nitric Oxide–Dependent and Nitric Oxide–Independent Transcriptional Responses to High Shear Stress in Endothelial Cells

Shear stress modulates gene expression in endothelial cells (ECs) partly through nitric oxide (NO), acting via enhanced cGMP formation by guanylyl cyclase (GC). We addressed non-cGMP–mediated transcriptional responses to shear stress in human umbilical ECs subjected to high-laminar shear stress (25 dyn/cm2; 150 minutes). RNA was isolated, reverse-transcribed, Cy3/5-labeled, and hybridized to 19 K human microarrays. High shear (n=6), high shear with 100 &mgr;mol/L l-NAME (n=3), and high shear with 10 &mgr;mol/L ODQ (GC inhibitor) in the perfusate (n=3) was compared with samples not subjected to flow. Among genes responding to high shear were HMOX1 (up) and PPARG (down). A high percentage of gene expression modulation by shear was absent during concomitant l-NAME or ODQ. Several transcriptional modulators were found (up: SOX5, SOX25, ZNF151, HOXD10; down: SOX11); a number of genes were regulated by shear and by shear with ODQ, but not regulated during l-NAME, indicating a nitric oxide synthase (NOS)–dependent, guanylyl cyclase (GC)–independent pathway. Several genes only responded to shear stress during l-NAME, others only responded to shear during ODQ. Upstream binding site analysis indicated shear stress and NO-dependent regulation of transcription via SOX5 and SOX9. Although NO importantly modulated the effect of shear stress on EC transcription, HMOX1 was consistently induced by shear stress, but not dependent on NOS or GC. Using bio-informatics software and databases, a promoter analysis identified SOX5 and SOX9 as potential, novel, shear-sensitive, and NO-dependent transcriptional regulators. The role of HMOX1 as a potential backup for NOS and the downstream role of SOXes should be explored.

Specific immuno capturing of the staphylococcal superantigen toxic-shock syndrome toxin-1 in plasma.

Toxic‐shock syndrome is primarily caused by the Toxic‐shock syndrome toxin 1 (TSST‐1), which is secreted by the Gram‐positive bacterium Staphylococcus aureus. The toxin belongs to a family of superantigens (SAgs) which exhibit several shared biological properties, including the induction of massive cytokine release and Vβ‐specific T‐cell proliferation. In this study we explored the possibility to use monoclonal Variable domains of Llama Heavy‐chain antibodies (VHH) in the immuno capturing of TSST‐1 from plasma. Data is presented that the selected VHHs are highly specific for TSST‐1 and can be efficiently produced in large amounts in yeast. In view of affinity chromatography, the VHHs are easily coupled to beads, and are able to deplete TSST‐1 from plasma at very low, for example, pathologically relevant, concentrations. When spiked with 4u2009ng/mL TSST‐1 more than 96% of TSST‐1 was depleted from pig plasma. These data pave the way to further explore application of high‐affinity columns in the specific immuno depletion of SAgs in experimental sepsis models and in sepsis in humans. Biotechnol. Bioeng. 2009; 104: 143–151

Role of Circulating Karyocytes in the Initiation and Progression of Atherosclerosis

Cardiovascular disease is still hard to predict in an individual. The main focus in cardiovascular research has been on endothelial cells and vascular smooth muscle cells of the vessel wall and their interactions with the blood flow. Alterations in the properties of the blood have received a lot of attention in biochemical terms. Interestingly, alterations in the properties of circulating cells have received less attention. We propose that presence of 1 or more risk factors together with normal physiological stimuli induce redox-dependent changes in leukocyte gene transcription with pathophysiological responses. Thus, risk factors render leukocytes hypersensitive to normal stimuli. Risk factors can be subdivided into physical and chemical factors. Superimposed on physiological regulators of leukocyte function, these risk factors promote a cellular pro-oxidative state. Redox-sensitive transcription factors are activated, leading to responses involving inflammation, adhesion, migration, and additional reactive oxygen species generation. As a consequence, monitoring of individual gene expression signatures of these cells could well increase our understanding of the mechanisms by which leukocytes and, in particular, monocytes function. Furthermore, transcriptomes of these cells could be used to investigate the aggressiveness of the atherosclerotic process or to guide treatment in the patient with risk factors for atherosclerosis.

Unchanged cardiac angiotensin II levels accompany losartan-sensitive cardiac injury due to nitric oxide synthase inhibition

Chronic nitric oxide synthase (NOS) inhibition results in hypertension and myocardial injury. In a rapid and severe model of chronic NOS inhibition, we determined the role of angiotensin II in these effects by using angiotensin II receptor blockade and by measuring cardiac angiotensin II concentrations before and during development of cardiac damage. Rats received either no treatment, the NOS inhibitor Nomega-nitro-L-arginine (L-NNA; 500 mg/l), the angiotensin AT(1) receptor antagonist losartan (400 mg/kg chow), or L-NNA plus losartan for 21 days. In the second protocol, five groups of rats received L-NNA (500 mg/l) for 0, 4, 7, 14 and 21 days, respectively. L-NNA increased systolic blood pressure (SBP) (227+/-8 versus 143+/-6 mm Hg; P<0.01), heart weight index (0.44+/-0.02 versus 0.32+/-0.01; P<0.01) and induced coronary vasculitis and myocardial necrosis. Co-treatment with losartan prevented all changes. L-NNA during 4 days decreased cardiac angiotensin II (23+/-4 versus 61+/-15 fmol/g; P<0.05). Although after 7 days, fresh infarcts and after 14 days organized infarcts were present, cardiac angiotensin II was only slightly increased after 21 days (100+/-10 fmol/g; P<0.05). In conclusion, losartan-sensitive cardiac damage due to chronic NOS inhibition is not associated with primary increase of cardiac angiotensin II, suggesting that chronic NOS inhibition increases cardiac sensitivity for angiotensin II.

Oleic acid increases mitochondrial reactive oxygen species production and decreases endothelial nitric oxide synthase activity in cultured endothelial cells

Elevated plasma levels of free fatty acids (FFA) are associated with increased cardiovascular risk. This may be related to FFA-induced elevation of oxidative stress in endothelial cells. We hypothesized that, in addition to mitochondrial production of reactive oxygen species, endothelial nitric oxide synthase (eNOS)-mediated reactive oxygen species production contributes to oleic acid (OA)-induced oxidative stress in endothelial cells, due to eNOS uncoupling. We measured reactive oxygen species production and eNOS activity in cultured endothelial cells (bEnd.3) in the presence of OA bound to bovine serum albumin, using the CM-H2DCFDA assay and the L-arginine/citrulline conversion assay, respectively. OA induced a concentration-dependent increase in reactive oxygen species production, which was inhibited by the mitochondrial complex II inhibitor thenoyltrifluoroacetone (TTFA). OA had little effect on eNOS activity when stimulated by a calcium-ionophore, but decreased both basal and insulin-induced eNOS activity, which was restored by TTFA. Pretreatment of bEnd.3 cells with tetrahydrobiopterin (BH4) prevented OA-induced reactive oxygen species production and restored inhibition of eNOS activity by OA. Elevation of OA levels leads to both impairment in receptor-mediated stimulation of eNOS and to production of mitochondrial-derived reactive oxygen species and hence endothelial dysfunction.

Normal TGF Responsiveness During Chronic Treatment With Angiotensin-Converting Enzyme Inhibition: Role of AT1 Receptors

Acute inhibition of angiotensin II formation by angiotensin-converting enzyme inhibition (ACE-I) attenuates tubuloglomerular feedback (TGF) responsiveness. This has been proposed to facilitate sodium excretion, which contributes to the antihypertensive effects of ACE-I. However, in previous experiments in spontaneously hypertensive Fawn-hooded rats, TGF responses were normal during chronic ACE-I treatment. In the present study, we investigated TGF responsiveness during chronic ACE-I treatment in normotensive rats and the involvement of changes in nitric oxide or angiotensin II activity. Maximum TGF responses were assessed in control Sprague-Dawley rats and in rats acutely (acute ACE-I, 3 &mgr;g/min IV) and chronically (chronic ACE-I, 100 mg/L PO 2 to 3 weeks+acute 3 &mgr;g/min enalaprilat IV) treated with ACE-I. In all groups, TGF responses were also assessed during late proximal tubular perfusion with 1 mmol/L nitro-l-arginine. In a last group, the chronic ACE-I treatment was combined with acute ACE-I and high doses of intrarenal losartan (acute 3 &mgr;g/min enalaprilat IV+50 mg/kg losartan). The maximum TGF responses in acutely treated ACE-I rats were strongly attenuated (0.7±0.4 mm Hg versus 6.5±0.8 mm Hg in control rats, P <0.05). Mean arterial pressure was lower in the chronically treated ACE-I group (107±5 mm Hg versus 126±5 mm Hg in control rats, P <0.05); however, TGF responses were normal (6.4±0.9 mm Hg). Intraluminal nitro-l-arginine infusion did not influence TGF responses during acute ACE-I (2.3±0.4 mm Hg) but enhanced TGF responses during chronic ACE-I to the same extent as in control rats (14.5±2.3 versus 16.7±1.9 mm Hg, NS). In the rats chronically treated with ACE-I with superimposed acute infusion of losartan or chronically treated with losartan, TGF responses were significantly attenuated (1.8±0.8 mm Hg and 2.6±0.8 mm Hg, respectively;P <0.05 versus chronic ACE-I and control). Prolonged administration with ACE-I is associated with normal TGF responses. This phenomenon appears to be mediated by AT1 receptors, because acute treatment with losartan in rats chronically treated with ACE-I and chronic treatment with losartan lead to strong attenuation of TGF responses.

Expression Profiling in Cardiovascular Disease Using Microarrays

This chapter is focused on the application of genome-wide screening techniques, with the emphasis on microarrays to assess gene expression and the application in cardiovascular research.Whywould one still want to look at gene expressionwhen proteomics is also available? One point of view is that gene expression analysis deals with the perceptive site of the cell: how do extracellular signals affect the program of the cell. How does induction of second messengers lead to alterations in gene expression? Another viewpoint is that without the transcript there is no protein. There are as many examples of good correlations between transcription and protein expression as there are exceptions; however, the no transcript, no protein concept remains of important value. Good examples of close relations between gene and protein expression are formed by the components of the renin– angiotensin system: mice with one to four copies of the human renin gene have proportional protein expression (Smithies et al., 2000). A more defensive point of view is that proteomics is still associated with major problems, in particular with respect to the activation state and modifications of proteins. Also, there is only one genome in, from which multiple transcriptomes are derived, depending upon the state of the cell. The number of possible proteomes in a cell is, however, breathtaking.