Anja Bondke Persson

Vascular growth in health and disease

Vascular growth forms the first functional organ system during development, and continues into adult life, wherein it is often associated with disease states. Genetically determined vasculogenesis produces a primary vascular plexus during ontogenesis. Angiogenesis, occurring, e.g., in response to metabolic stress within hypoxic tissues, enhances tissue capillarization. Arteriogenesis denotes the adaptive outgrowth of pre-existent collateral arteries to bypass arterial stenoses in response to hemodynamic changes. It has been debated whether vasculogenesis occurs in the adult, and whether or not circulating progenitor cells structurally contribute to vessel regeneration. Secondly, the major determinants of vascular growth – genetic predisposition, metabolic factors (hypoxia), and hemodynamics – cannot be assigned in a mutually exclusive fashion to vasculogenesis, angiogenesis, and arteriogenesis, respectively; rather, mechanisms overlap. Lastly, all three mechanisms of vessel growth seem to contribute to physiological embryogenesis as well as adult adaptive vascularization as occurs in tumors or to circumvent arterial stenosis. Thus, much conceptual and terminological confusion has been created, while therapies targeting neovascularization have yielded promising results in the lab, but failed randomized studies when taken to the bedside. Therefore, this review article aims at providing an exact definition of the mechanisms of vascular growth and their contribution to embryonic development as well as adult adaptive revascularization. We have been looking for potential reasons for why clinical trials have failed, how vitally the application of appropriate methods of measuring and assessment influences study outcomes, and how relevant, e.g., results gained in models of vascular occlusive disease may be for antineoplastic strategies, advocating a reverse bedside-to-bench approach, which may hopefully yield successful approaches to therapeutically targeting vascular growth.

Iodinated contrast media cause endothelial damage leading to vasoconstriction of human and rat vasa recta

Contrast-induced acute kidney injury is an important clinical event with a worldwide increasing number of cases. Medullary hypoperfusion and hypoxia due to constriction of vasa recta are main factors in the pathophysiology of acute kidney injury. However, the mechanism of contrast media (CM)-induced vessel constriction is not known. We tested the hypothesis that vasa recta constriction is a consequence of endothelial dysfunction due to the cytotoxicity of CM. Human and rat descending vasa recta (DVR) were isolated and perfused with CM, and the luminal diameter was analyzed. For morphological analysis of the endothelium, renal arteries were CM perfused and then processed for electron microscopy. Transcellular electrical resistance was used to estimate CM-induced changes in the permeability of human umbilical vein endothelial cell (HUVEC) layers. Perfusion with CM constricted human and rat DRV (to 54.3 and 50.9% of initial diameter, respectively). This was blunted by adrenomedullin (77.7 and 77.1%, respectively). The ANG II response was enhanced by CM in rat DVR (reduction to 15.6 and 35.0% of initial diameter, respectively). Adrenomedullin blunted this effect (67.5%). CM led to endothelial damage of renal arteries characterized by a ragged surface, with sharply protruding intimal folds, spindle-like shape, and bulging in the lumen. These phenomena were reduced by adrenomedullin. The permeability of HUVEC cell layers was increased by CM, and this went along with increased myosin light chain phosporylation. Again, adremonedullin reduced the CM effect. Our study suggests that the constrictor effect of CM on the renal medullary microvasculature is a consequence of endothelial cell damage and the resulting endothelial dysfunction.

Granulocyte Colony-Stimulating Factor Improves Cerebrovascular Reserve Capacity by Enhancing Collateral Growth in the Circle of Willis

Background and Purpose: Restoration of cerebrovascular reserve capacity (CVRC) depends on the recruitment and positive outward remodeling of preexistent collaterals (arteriogenesis). With this study, we provide functional evidence that granulocyte colony-stimulating factor (G-CSF) augments therapeutic arteriogenesis in two animal models of cerebral hypoperfusion. We identified an effective dosing regimen that improved CVRC and stimulated collateral growth, thereby improving the outcome after experimentally induced stroke. Methods: We used two established animal models of (a) cerebral hypoperfusion (mouse, common carotid artery ligation) and (b) cerebral arteriogenesis (rat, 3-vessel occlusion). Following therapeutic dose determination, both models received either G-CSF, 40 µg/kg every other day, or vehicle for 1 week. Collateral vessel diameters were measured following latex angiography. Cerebrovascular reserve capacities were assessed after acetazolamide stimulation. Mice with left common carotid artery occlusion (CCAO) were additionally subjected to middle cerebral artery occlusion, and stroke volumes were assessed after triphenyltetrazolium chloride staining. Given the vital role of monocytes in arteriogenesis, we assessed (a) the influence of G-CSF on monocyte migration in vitro and (b) monocyte counts in the adventitial tissues of the growing collaterals in vivo. Results: CVRC was impaired in both animal models 1 week after induction of hypoperfusion. While G-CSF, 40 µg/kg every other day, significantly augmented cerebral arteriogenesis in the rat model, 50 or 150 µg/kg every day did not show any noticeable therapeutic impact. G-CSF restored CVRC in mice (5 ± 2 to 12 ± 6%) and rats (3 ± 4 to 19 ± 12%). Vessel diameters changed accordingly: in rats, the diameters of posterior cerebral arteries (ipsilateral: 209 ± 7–271 ± 57 µm; contralateral: 208 ± 11–252 ± 28 µm) and in mice the diameter of anterior cerebral arteries (185 ± 15–222 ± 12 µm) significantly increased in the G-CSF groups compared to controls. Stroke volume in mice (10 ± 2%) was diminished following CCAO (7 ± 4%) and G-CSF treatment (4 ± 2%). G-CSF significantly increased monocyte migration in vitro and perivascular monocyte numbers in vivo. Conclusion: G-CSF augments cerebral collateral artery growth, increases CVRC and protects from experimentally induced ischemic stroke. When comparing three different dosing regimens, a relatively low dosage of G-CSF was most effective, indicating that the common side effects of this cytokine might be significantly reduced or possibly even avoided in this indication.

Acetylsalicylic acid, but not clopidogrel, inhibits therapeutically induced cerebral arteriogenesis in the hypoperfused rat brain.

This study investigated the effects of acetylsalicylic acid (ASA) and clopidogrel, standardly used in the secondary prevention of vascular occlusions, on cerebral arteriogenesis in vivo and in vitro. Cerebral hypoperfusion was induced by three-vessel occlusion (3-VO) in rats, which subsequently received vehicle, ASA (6.34 mg/kg), or clopidogrel (10 mg/kg). Granulocyte colony-stimulating factor (G-CSF), which enhanced monocyte migration in an additional cell culture model, augmented cerebrovascular arteriogenesis in subgroups (40 μg/kg). Cerebrovascular reactivity and vessel diameters were assessed at 7 and 21 days. Cerebrovascular reserve capacity was completely abolished after 3-VO and remained severely compromised after 7 (−14 ± 14%) and 21 (−5 ± 11%) days in the ASA groups in comparison with controls (4 ± 5% and 10 ± 10%) and clopidogrel (4 ± 13% and 10 ± 8%). It was still significantly decreased when ASA was combined with G-CSF (1 ± 4%) compared with G-CSF alone (20 ±8%). Posterior cerebral artery diameters confirmed these data. Monocyte migration into the vessel wall, improved by G-CSF, was significantly reduced by ASA. Acetylsalicylic acid, but not clopidogrel, inhibits therapeutically augmented cerebral arteriogenesis.

Multilevel regulation of HIF-1 signaling by TTP

Phosphorylation of the RNA-binding protein tristetraprolin (TTP) by p38 MAPK/MK2 does not prevent its RNA interaction and switches the mode of TTP action from destabilization to stabilization of the HIF-1α mRNA and subsequent activation of HIF-1 signaling.

Wilms’ tumor protein Wt1 regulates the Interleukin-10 (IL-10) gene

We identified the Wilms’ tumor protein, Wt1, as a novel transcriptional activator of the immunosuppressant cytokine interleukin‐10 (IL‐10). Silencing of Wt1 by RNA interference reduced IL‐10 mRNA levels by approximately 90%. IL‐10 transcripts were increased more than 15‐fold upon forced expression of Wt1. Electrophoretic mobility shift assay and chromatin immunoprecipitation revealed a cis‐element that was responsible for activation of the IL‐10 promoter by Wt1 in murine macrophages. Mutation of the Wt1 binding motif abrogated stimulation of the IL‐10 promoter by tumor necrosis factor‐α (TNFα). These results suggest a novel immune regulatory function of Wt1 in controlling IL‐10 gene expression.

Hypoxia-induced gene expression results from selective mRNA partitioning to the endoplasmic reticulum

Protein synthesis is a primary energy-consuming process in the cell. Therefore, under hypoxic conditions, rapid inhibition of global mRNA translation represents a major protective strategy to maintain energy metabolism. How some mRNAs, especially those that encode crucial survival factors, continue to be efficiently translated in hypoxia is not completely understood. By comparing specific transcript levels in ribonucleoprotein complexes, cytoplasmic polysomes and endoplasmic reticulum (ER)-bound ribosomes, we show that the synthesis of proteins encoded by hypoxia marker genes is favoured at the ER in hypoxia. Gene expression profiling revealed that transcripts particularly increased by the HIF-1 transcription factor network show hypoxia-induced enrichment at the ER. We found that mRNAs favourably translated at the ER have higher conservation scores for both the 5′- and 3′-untranslated regions (UTRs) and contain less upstream initiation codons (uAUGs), indicating the significance of these sequence elements for sustained mRNA translation under hypoxic conditions. Furthermore, we found enrichment of specific cis-elements in mRNA 5′- as well as 3′-UTRs that mediate transcript localization to the ER in hypoxia. We conclude that transcriptome partitioning between the cytoplasm and the ER permits selective mRNA translation under conditions of energy shortage.

Amine oxidase copper containing 1 (AOC1) is a downstream target gene of the Wilms tumor protein, WT1, during kidney development

Background: Polyamines and their diamine precursor putrescine are ubiquitous organic polycations involved in cell growth and proliferation. Results: The Wilms tumor suppressor, WT1, stimulates transcription of the AOC1 gene, which encodes the key enzyme for putrescine breakdown. Conclusion: WT1-dependent regulation of putrescine degradation, mediated by AOC1, has a role in kidney morphogenesis. Significance: The findings provide novel insights into transcriptional mechanisms controlling genitourinary development. Amine oxidase copper-containing 1 (AOC1; formerly known as amiloride-binding protein 1) is a secreted glycoprotein that catalyzes the degradation of putrescine and histamine. Polyamines and their diamine precursor putrescine are ubiquitous to all organisms and fulfill pivotal functions in cell growth and proliferation. Despite the importance of AOC1 in regulating polyamine breakdown, very little is known about the molecular mechanisms that control its expression. We report here that the Wilms tumor protein, WT1, which is necessary for normal kidney development, activates transcription of the AOC1 gene. Expression of a firefly luciferase reporter under control of the proximal AOC1 promoter was significantly enhanced by co-transfection of a WT1 expression construct. Binding of WT1 protein to a cis-regulatory element in the AOC1 promoter was confirmed by electrophoretic mobility shift assay and chromatin immunoprecipitation. Antisense inhibition of WT1 protein translation strongly reduced Aoc1 transcripts in cultured murine embryonic kidneys and gonads. Aoc1 mRNA levels correlated with WT1 protein in several cell lines. Double immunofluorescent staining revealed a co-expression of WT1 and AOC1 proteins in the developing genitourinary system of mice and rats. Strikingly, induced changes in polyamine homeostasis affected branching morphogenesis of cultured murine embryonic kidneys in a developmental stage-specific manner. These findings suggest that WT1-dependent control of polyamine breakdown, which is mediated by changes in AOC1 expression, has a role in kidney organogenesis.

Keeping in synch.

Have you ever felt socially jet lagged? Running out of time? Or that everyone around you just keeps running late? If the answer is yes, this might not only be your subjective feeling, but rather a measurable outcome in a society comprising young and old, owls and larks, and all of them with clocks running in different periods, phases and amplitudes. This article is protected by copyright. All rights reserved.

Induction of Cerebral Arteriogenesis in Mice

Unilateral common carotid artery occlusion (CCAO) is a standardized method to initiate collateral artery growth (arteriogenesis) in mouse brain. After CCAO is induced, blood circulation in the circle of Willis is changed and increases shear stress, which triggers increased arterial diameter and improvements in cerebrovascular reserve capacity. Functional improvement can be quantified after experimentally induced stroke by external middle cerebral artery occlusion (MCAO). Stroke volume is evaluated by standard tetrazolium chloride (TTC) staining. Here, we describe in vivo methods of CCAO and MCAO in detail and also the evaluation of stroke volume by TTC staining.