Involvement of neuronal NO synthase in collateral artery growth.

To evaluate the role of neuronal nitric oxides synthase (nNOS) in collateral artery growth (arteriogenesis), we analyzed the expression pattern of nNOS at distinct time points on RNA and protein levels in a rabbit and a murine model of peripheral arteriogenesis. In the rabbit model, Northern blot analyses revealed a significant upregulation of nNOS at 6 h (1.6-fold), 12 h (2.2-fold) and 24 h (2.0-fold) after induction of arteriogenesis via femoral artery ligation, when compared to the sham operated side. In mice, an upregulation of nNOS was also detected using Northern blot (at 6 h, 12 h) and qRT-PCR (12 h: 2.4-fold). On the protein level, nNOS was found to be upregulated 24 h after femoral artery ligation. Immunohistochemical staining showed that nNOS was localized in endothelial and smooth muscle cells of collateral arteries, as well as in skeletal muscle and nerves. In summary, our data provide evidence that nNOS is not constitutively expressed, but is induced during arteriogenesis, playing a role in supplying reactive oxygen species such as H2O2 and low levels of NO.

Effect of intermittent high altitude hypoxia on gene expression in rat heart and lung.

Hypoxia has been identified as an important stimulus for gene expression during embryogenesis and in various pathological situations. Its influence under physiological conditions, however, has only been studied occasionally. We therefore investigated the effect of intermittent high altitude hypoxia on the mRNA expression of different cytokines and protooncogenes, but also of other genes described to be regulated by hypoxia, in the left ventricle (LV), the right ventricle (RV), atria and the lung of adult rats after simulation of hypoxia in a barochamber (5000 m, 4 hours to 10 days). Heme oxygenase-1 as well as transforming growth factor-beta1 showed an increased expression in all regions of the heart and the lung at different periods of hypoxia. For lactate dehydrogenase-A, we found a significant up-regulation in the RV and the lung, for lactate dehydrogenase-B up-regulation in the RV, but down-regulation in the LV and the atria. Vascular endothelial growth factor was up-regulated in the RV, the LV and the lung, but down-regulated in the atria. Its receptor Flk-1 mRNA was significantly increased in the atria and RV only. Expression of c-fos was found in the LV and RV only after 4 hours of hypoxia. The level of c-jun was significantly increased in the LV but decreased in the atria. Our data clearly demonstrate that intermittent hypoxia is a modulator of gene expression under physiological conditions. It differently regulates the expression of distinct genes not only in individual organs but even within one organ, i.e. in the heart.

Receptor-independent role of the urokinase-type plasminogen activator during arteriogenesis.

To define the role of the plasminogen activators (PAs) urokinase PA (uPA) and tissue PA (tPA) as well as the uPA receptor (uPAR) in arteriogenesis, we investigated their impact in a rabbit and mouse model of adaptive collateral artery growth. Collateral artery growth was induced by occlusion of the femoral artery in rabbit and wild-type (WT) mice and in mice with targeted inactivation of uPA (uPA-/-), tPA (tPA-/-), or uPAR (uPAR-/-). Northern blot results revealed a significant up-regulation of uPA but not uPAR or tPA in the early phase of arteriogenesis in rabbit and WT mice. This up-regulation on RNA level was followed by an increased protein level and enzymatic activity. Impaired perfusion recovery upon femoral artery ligation was observed by laser Doppler analysis in vivo in uPA-deficient mice but not in uPAR or tPA deficiency compared with WT mice. Immunohistochemical studies revealed an association of leukocyte infiltration with arteriogenesis in WT mice that was strongly reduced in uPA-/- but not in uPAR- or tPA-deficient mice. We conclude that arteriogenesis is promoted by an uPA-mediated infiltration of leukocytes that is not dependent on uPAR.

Role of ischemia and of hypoxia-inducible genes in arteriogenesis after femoral artery occlusion in the rabbit.

Vascular endothelial growth factor (VEGF) is known to play an important role in angiogenesis. Its place in collateral artery growth (arteriogenesis), however, is still debated. In the present study, we analyzed the expression of VEGF and its receptors (Flk-1 and Flt-1) in a rabbit model of collateral artery growth after femoral artery occlusion. Hypoxia presents the most important stimulus for VEGF expression. We therefore also investigated the expression level of distinct hypoxia-inducible genes (HIF-1alpha, LDH A) and determined metabolic intermediates indicative for ischemia (ATP, creatine phosphate, and their catabolites). We found that arteriogenesis was not associated with an increased expression of VEGF or the mentioned hypoxia-inducible genes. Furthermore, the high-energy phosphates and their catabolites were entirely within normal limits. Despite the absence of an increased expression of VEGF and its receptors, collateral vessels increased their diameter by a factor of 10. The speed of collateral development could be increased by infusion of the chemoattractant monocyte chemotactic protein-1 but not by infusion of a 30 times higher concentration of VEGF. From these data, we conclude that under nonischemic conditions, arteriogenesis is neither associated with nor inducible by increased levels of VEGF and that VEGF is not a natural agent to induce arteriogenesis in vivo.

Ultrastructure and molecular histology of rabbit hind-limb collateral artery growth (arteriogenesis).

Previous studies in the canine heart had shown that the growth of collateral arteries occurs via proliferative enlargement of pre-existing arteriolar connections (arteriogenesis). In the present study, we investigated the ultrastructure and molecular histology of growing and remodeling collateral arteries that develop after femoral artery occlusion in rabbits as a function of time from 2 h to 240 days after occlusion. Pre-existent arteriolar collaterals had a diameter of about 50 microm. They consisted of one to two layers of smooth muscle cells (SMCs) and were morphologically indistinguishable from normal arterioles. The stages of arteriogenesis consisted of arteriolar thinning, followed by transformation of SMCs from the contractile- into the proliferative- and synthetic phenotype. Endothelial cells (ECs) and SMCs proliferated, and SMCs migrated and formed a neo-intima. Intercellular adhesion molecule (ICAM-1) and vascular cell adhesion molecule (VCAM-1) showed early upregulation in ECs, which was accompanied by accumulation of blood-derived macrophages. Mitosis of ECs and SMCs started about 24 h after occlusion, whereas adhesion molecule expression and monocyte adhesion occurred as early as 12 h after occlusion, suggesting a role of monocytes in vascular cell proliferation. Treatment of rabbits with the pro-inflammatory cytokine MCP-1 increased monocyte adhesion and accelerated vascular remodeling. In vitro shear-stress experiments in cultured ECs revealed an increased phosphorylation of the focal contacts after 30 min and induction of ICAM-1 and VCAM-1 expression between 2 h and 6 h after shear onset, suggesting that shear stress may be the initiating event. We conclude that the process of arteriogenesis, which leads to the positive remodeling of an arteriole into an artery up to 12 times its original size, can be modified by modulators of inflammation.

Elevated levels of endogenous adenosine alter metabolism and enhance reduction in contractile function during low-flow ischemia: associated changes in expression of Ca(2+)-ATPase and phospholamban.

Adenosine has several potentially cardioprotective effects including vasodilatation, reduction in heart rate and alterations in metabolism. Adenosine inhibits catecholamine-induced increase in contractile function mainly through inhibition of phosphorylation of phospholamban (PLB), the main regulatory protein of Ca(2+)-ATPase in sarcoplasmic reticulum (SR), and during ischemia it reduces calcium (Ca2+) overload. In this study we examined the effects of endogenous adenosine on contractile function and metabolism during low-flow ischemia (LFI) and investigated whether endogenous adenosine can alter expression of the Ca(2+)-ATPase/PLB-system and other Ca(2+)-regulatory proteins. Isolated blood-perfused piglet hearts underwent 120 min 10% flow. Hearts were treated with either saline, the adenosine receptor blocker (8)-sulfophenyl theophylline (8SPT, 300 micromol/l) or the nucleoside transport inhibitor draflazine (1 micromol/l). During LFI, 8SPT did not substantially influence metabolic or functional responses. However, draflazine enhanced the reduction in heart rate, contractile force and MVO(2), with less release of H+ and CO2. Before LFI there were no significant differences between groups for any of the proteins (Ca(2+)-ATPase, ryanodine-receptor, Na+/K(+)-ATPase) or mRNAs (Ca(2+)-ATPase, PLB, calsequestrin, Na+/Ca(2+)-exchanger) measured. At end of LFI mRNA-level of PLB was higher in draflazine-treated hearts compared to both other groups (P<0.01 vs both). Also, at end of LFI protein-level of Ca(2+)-ATPase was lower in draflazine-treated hearts (P<0.05 vs both), and a parallel trend towards a lower mRNA-level was seen (P=0.11 vs saline and P=0.43 vs 8SPT). During LFI tissue Ca2+ tended to rise in saline- and 8SPT-treated hearts but not in draflazine-treated hearts (at end of LFI, P=0.01 vs 8SPT). We conclude that the amount of adenosine normally produced during LFI does not substantially influence function and metabolism. However, increased endogenous levels by draflazine enhance downregulation of function and reduce signs of anaerobic metabolism. At end of LFI associated changes in expression of PLB and Ca(2+)-ATPase were seen. The functional significance was not determined in the present study. However, altered protein-levels might influence Ca(2+)-handling in sarcoplasmic reticulum and thus affect contractile force and tolerance to ischemia.

Gene expression after short periods of coronary occlusion.

Brief periods of coronary occlusion render the affected myocardium more tolerant to the otherwise devastating effects of long coronary occlusion. Besides this phenomena, called ischemic preconditioning, short periods of ischemia cause a regional dysfunction, namely myocardial stunning. The molecular mechanisms of both syndromes are not very well understood. We therefore investigated the expression of genes which may be involved in cardioprotection or repair processes. Using our porcine model of ischemia and reperfusion we were able to show an induction of genes coding for transcription factors (proto-oncogenes), for proteins involved in repair processes (heat shock genes), for proteins implicated in the calcium homeostasis (calcium-handling genes) and for growth factors. We could show that the increased mRNA levels are due to an enhanced transcriptional activity and not to a prolonged half-life of the transcripts. The angiogenic growth factor vascular endothelial growth factor (VEGF) represents an exception. It exhibits--in addition to a HIF-motif (Hypoxia Inducible Factor) in its promoter/enhancer--a protein binding region in its 3' UTR which when occupied renders the mRNA more stable. However to what extent the expression of the distinct genes contributes to the cardioprotective effect of ischemic preconditioning or myocardial stunning can only be presumed. Increased mRNA stability can be confered via adenosine which is produced during ischemia by ATP-breakdown. The demasking of unknown genes--via differential display reverse transcription polymerase chain reaction (DDRT-PCR)--should provide a more comprehensive view of the mechanisms underlying both processes.

A splice hepadnavirus RNA that is essential for virus replication.

According to the current model of hepadnavirus gene expression, the viral envelope proteins are produced from unspliced subgenomic RNAs, in contrast to the retroviral mechanism, where the subgenomic env RNA is generated by RNA splicing. We now describe and characterize a novel duck hepatitis B virus RNA species which is derived from the RNA pregenome by loss of a 1.15 kb intron. This RNA (termed spliced L RNA) codes for the large surface protein (L protein), as does the previously described unspliced mRNA (the preS RNA); however, it differs in 5' leader sequence and promoter control. Mutational analysis indicates that the spliced L RNA is functionally important for virus replication in infected hepatocytes and ducks, but not for virus formation from transfected DNA genomes. This suggests that the newly discovered second pathway for L protein synthesis plays a distinct role in an early step in the viral life cycle.