Philipp Hillmeister

Pulsatile shear and Gja5 modulate arterial identity and remodeling events during flow-driven arteriogenesis

In the developing chicken embryo yolk sac vasculature, the expression of arterial identity genes requires arterial hemodynamic conditions. We hypothesize that arterial flow must provide a unique signal that is relevant for supporting arterial identity gene expression and is absent in veins. We analyzed factors related to flow, pressure and oxygenation in the chicken embryo vitelline vasculature in vivo. The best discrimination between arteries and veins was obtained by calculating the maximal pulsatile increase in shear rate relative to the time-averaged shear rate in the same vessel: the relative pulse slope index (RPSI). RPSI was significantly higher in arteries than veins. Arterial endothelial cells exposed to pulsatile shear in vitro augmented arterial marker expression as compared with exposure to constant shear. The expression of Gja5 correlated with arterial flow patterns: the redistribution of arterial flow provoked by vitelline artery ligation resulted in flow-driven collateral arterial network formation and was associated with increased expression of Gja5. In situ hybridization in normal and ligation embryos confirmed that Gja5 expression is confined to arteries and regulated by flow. In mice, Gja5 (connexin 40) was also expressed in arteries. In the adult, increased flow drives arteriogenesis and the formation of collateral arterial networks in peripheral occlusive diseases. Genetic ablation of Gja5 function in mice resulted in reduced arteriogenesis in two occlusion models. We conclude that pulsatile shear patterns may be central for supporting arterial identity, and that arterial Gja5 expression plays a functional role in flow-driven arteriogenesis.

Induction of cerebral arteriogenesis leads to early-phase expression of protease inhibitors in growing collaterals of the brain

Cerebral arteriogenesis constitutes a promising therapeutic concept for cerebrovascular ischaemia; however, transcriptional profiles important for therapeutic target identification have not yet been investigated. This study aims at a comprehensive characterization of transcriptional and morphologic activation during early-phase collateral vessel growth in a rat model of adaptive cerebral arteriogenesis. Arteriogenesis was induced using a three-vessel occlusion (3-VO) rat model of nonischaemic cerebral hypoperfusion. Collateral tissue from growing posterior cerebral artery (PCA) and posterior communicating artery (Pcom) was selectively isolated avoiding contamination with adjacent tissue. We detected differential gene expression 24 h after 3-VO with 164 genes significantly deregulated. Expression patterns contained gene transcripts predominantly involved in proliferation, inflammation, and migration. By using scanning electron microscopy, morphologic activation of the PCA endothelium was detected. Furthermore, the PCA showed induced proliferation (PCNA staining) and CD68+ macrophage staining 24 h after 3-VO, resulting in a significant increase in diameter within 7 days after 3-VO, confirming the arteriogenic phenotype. Analysis of molecular annotations and networks associated with differentially expressed genes revealed that early-phase cerebral arteriogenesis is characterised by the expression of protease inhibitors. These results were confirmed by quantitative real-time reverse transcription-PCR, and in situ hybridisation localised the expression of tissue inhibitor of metalloproteinase-1 (TIMP-1) and kininogen to collateral arteries, showing that TIMP-1 and kininogen might be molecular markers for early-phase cerebral arteriogenesis.

The Kallikrein–Kinin system

Pre-dating Hippocrates, more than 4000 years ago, human urine was already a fascinating research medium, climaxing in the renaissance. An oft-repeated story in the literature is that of a woman wishing to avoid a pregnancy diagnosis by blending her urine with that of a cow. Very much to her distress, the leche announced that both she and her cow were pregnant (Armstrong 2007)! It was in 1909 that Abelous and Bardier discovered in human urine a blood pressure-lowering substance (Abelous & Bardier 1909). In 1920, Pribram and Herrnheiser analysed the nondialysable fraction of human urine and injected it into a rabbit, which resulted in a marked fall in blood pressure (Pribram & Herrnheiser 1920). Six years later, Frey confirmed the effect of a hypotensive substance recovered from urine (Frey 1926). Frey and his co-worker Kraut believed the vasodilator substance extracted from urine was of pancreatic origin and named it kallikrein, derived from the Greek word for pancreas (Kraut & Werle 1930). Kallikreins are a heterogeneous group of serine proteases, which occur in the plasma as well as in several tissues, such as pancreas, kidney, intestine and salivary glands (Schachter 1979). A century of research later, we know that kallikreins are the initating element of the kallikrein–kinin system (KKS), cleaving the protein substrate kininogen to liberate kinins, among which the muscle-contracting nonapeptide bradykinin is known best (Bader 2009). Kinin acts mainly via two membrane receptors, the bradykinin receptor B1R and bradykinin receptor B2R, and this set of molecules (Kallikrien, substrate Kininogen, kinins and receptors) comprises the KKS, which seemed at first – based on the origin of its discovery – to be particularly relevant for renal function. Webster, Gilmore and Gill established the natriuretic/diuretic capabilities of kinins (Webster & Gilmore 1964, Gill et al. 1965). Bradykinin administration can increase renal blood flow without significantly changing glomerular filtration. Kinins reduce vascular resistance and stimulate water and electrolyte (sodium) excretion as a function of increased renal blood flow (Stein et al. 1972). Urine volume increases with a strong increase in fluid disposal to the distal nephron (Nasjletti & Malik 1981). (Terragno et al. 1972) and (McGiff et al. 1972), discovered that bradykinin stimulates prostaglandin release which led to the proposal that the kallikrein–kinin and arachidonate-prostaglandin systems are related (Fig. 1). Nowadays, an enormous amount of data has been published about the KKS, an important but still poorly understood and underestimated player in many different aspects (Leeb-Lundberg et al. 2005). A repertory of transgenic animals shed light onto the diverse functions of the KKS (Pesquero & Bader 2006). Besides its role in renal physiology, the KKS is relevant for cardiovascular function, such as blood pressure regulation (vasodilation), and has been implicated the pathogenesis of hypertension (Marceau & Regoli 2004, Regoli et al. 2012). Furthermore, the KKS is a central regulator of inflammatory processes and is in turn regulated by components of the innate immune response (Kaplan & Ghebrehiwet 2010). Various articles from Acta Physiologica (Oxford) have aided our understanding of the KKS’ major sites of action, that

Arteriogenesis Is Modulated By Bradykinin Receptor Signaling

Rationale: Positive outward remodeling of pre-existing collateral arteries into functional conductance arteries, arteriogenesis, is a major endogenous rescue mechanism to prevent cardiovascular ischemia. Collateral arterial growth is accompanied by expression of kinin precursor. However, the role of kinin signaling via the kinin receptors (B1R and B2R) in arteriogenesis is unclear. Objective: The purpose of this study was to elucidate the functional role and mechanism of bradykinin receptor signaling in arteriogenesis. Methods and Results: Bradykinin receptors positively affected arteriogenesis, with the contribution of B1R being more pronounced than B2R. In mice, arteriogenesis upon femoral artery occlusion was significantly reduced in B1R mutant mice as evidenced by reduced microspheres and laser Doppler flow perfusion measurements. Transplantation of wild-type bone marrow cells into irradiated B1R mutant mice restored arteriogenesis, whereas bone marrow chimeric mice generated by reconstituting wild-type mice with B1R mutant bone marrow showed reduced arteriogenesis after femoral artery occlusion. In the rat brain 3-vessel occlusion arteriogenesis model, pharmacological blockade of B1R inhibited arteriogenesis and stimulation of B1R enhanced arteriogenesis. In the rat, femoral artery ligation combined with arterial venous shunt model resulted in flow-driven arteriogenesis, and treatment with B1R antagonist R715 decreased vascular remodeling and leukocyte invasion (monocytes) into the perivascular tissue. In monocyte migration assays, in vitro B1R agonists enhanced migration of monocytes. Conclusions: Kinin receptors act as positive modulators of arteriogenesis in mice and rats. B1R can be blocked or therapeutically stimulated by B1R antagonists or agonists, respectively, involving a contribution of peripheral immune cells (monocytes) linking hemodynamic conditions with inflammatory pathways.

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.

Acute Physical Exercise and Long‐Term Individual Shear Rate Therapy Increase Telomerase Activity in Human Peripheral Blood Mononuclear Cells

Physical activity is a potent way to impede vascular ageing. However, patients who suffer from peripheral artery disease (PAD) are often unable to exercise adequately. For those patients, we have developed individual shear rate therapy (ISRT), which is an adaptation of external counterpulsation and enhances endovascular fluid shear stress to increase collateral growth (arteriogenesis). To evaluate the effects of physical exercise and ISRT on the telomere biology of peripheral blood mononuclear cells (PBMCs), we conducted two clinical trials.

PCSK9 regulates the chemokine receptor CCR2 on monocytes

Monocyte migration is a key element in atherosclerosis. LDL-C facilitates monocyte migration via induction of CCR2. PCSK9 regulates cell surface expression of the LDL-R and is expressed in vascular smooth muscle cells (VSMCs). The present study was done to investigate the regulation of PCSK9 in VSMCs and its impact on monocyte function. METHODS AND RESULTS PCSK9 mRNA and protein levels were upregulated in VSMCs by the TLR-4 ligand LPS, whereas TGF-β or angiotensin II had no effect. Induction of PCSK9 was selectively inhibited by TLR-4 blockade and further downstream by the SAPK/JNK-inhibitor SP600125, whereas inhibitors of ERK1/2, p38 or PI3-kinase pathways had no effect. Incubation of monocytes in conditioned media from LPS-stimulated VSMCs resulted in a significant reduction of LDL-R levels on monocytes, comparable to the effects of recombinant PCSK9. LDL-C increased monocyte CCR2 expression, which augmented monocyte migration towards MCP-1. This LDL-C dependent monocyte chemotaxis was inhibited by supernatants from LPS-stimulated VSMCs, similar to recombinant PCSK9 and a specific LDL-R blocking antibody. CONCLUSION PCSK9 is regulated in VSMCs by TLR-4 - SAPK/JNK signaling, a pathway important in inflammation and metabolism. VSMC-derived PCSK9 reduces monocyte LDL-R expression, affecting LDL-C/LDL-R-mediated CCR2-expression on monocytes, which is crucial to cell motility and atherogenesis.

Leucocyte telomere length as marker for cardiovascular ageing.

Already, the ancient Greeks established theories to explain unusual longevity. Herodotus, an ancient Greek historian, mentions a fountain of youth to explain the remarkably long-life expectancy (120 years) of the Macrobian people (Herodotus 450– 420 BC) (Fig. 1). The underlying question remained the same over the centuries, although it is nowadays addressed with scientific means as, for example, in the New England Centenarian Study (Sebastiani & Perls 2012). However, a ‘universal youth code’ remains to be found. This review focuses on recent progress in understanding how the immune system ages and how it thereby influences the world’s No 1 cause of mortality: cardiovascular disease (WHO 2013). To understand how the ageing of the immune system can be measured, it is necessary to first analyse the most frequently assessed ageing parameter: telomere length of circulating leucocytes. Telomeres are non-coding, repetitive base sequences (50-TTAGGG n30) at the ends of linear chromosomes, protecting the chromosomes from nuclease-mediated degradation and end-to-end fusion (Blackburn 2001). When a cell divides, it needs to replicate its full genomic information coded in the DNA. But during mitosis, the end of the lagging strand cannot be replicated entirely by the DNA polymerase, which causes the so-called end-replication problem (Levy et al. 1992). In consequence, telomeres shorten with every cell division until the cell goes into cell cycle arrest (cellular senescence) (Blackburn 2000). Thereby, the telomere length defines the number of possible cell divisions for somatic cells (Allsopp et al. 1992). Of course, germline cells, for example, need stable chromosomes to pass on genetic information from generation to generation. For this purpose, they express an enzyme called telomerase. The telomerase adds telomeric repeats to the singlestranded overhang and thus counteracts telomere shortening, offering the potential to divide limitlessly (Greider & Blackburn 1985) (Fig. 2). Telomere length in circulating leucocytes (LTL) correlates negatively with the chronological age of an individual (Iwama et al. 1998). More importantly, LTL can be altered by various environmental and genetic factors, which are known to be relevant for biological ageing (Mandal a et al. 2012, van den Munckhof et al. 2012). Recent findings suggested a major influence of oxidative stress and chronic inflammation on LTL (von Zglinicki 2002, Demissie et al. 2006, Houben et al. 2008, O’Donovan et al. 2011, Saliques et al. 2011). In this context, Aviv considers the telomeres of leucocytes to be ‘a record of the cumulative burden of inflammation and oxidative stress’ (Aviv 2009). Oxidative stress and chronic inflammation link LTL to the pathogenesis of cardiovascular diseases (CVD), because oxidative stress and chronic inflammation play a major role in development of CVD and its risk factors (Haines et al. 2012, Park et al. 2012, Carlstr€ om et al. 2013, Van e ckov a et al. 2013). Another relation is provided by the leucocytes themselves, because they mediate formation of atherosclerotic plaques: transformed macrophages, so-called ‘foam cells’, internalize oxLDL and release multiple cytokines (Brown & Goldstein 1990, Moore et al. 2013). The ongoing inflammatory process recruits leucocytes, which are replaced by augmented proliferative activity of hematopoietic stem cells in the bone marrow. As hematopoietic stem cells have only insufficient telomerase activity, they are unable to compensate for accelerated telomere shortening (Engelhardt et al. 1997). Consequently, atherosclerotic plaque formation contributes to hematopoietic stem cell ageing, reflected by decreased telomere length of their daughter cells: circulating leucocytes. The most important risk factors for cardiovascular disease are smoking, diabetes mellitus, hypertension, obesity, a sedentary lifestyle and psychosocial problems (Yusuf et al. 2004). Except for psychosocial issues, they all promote oxidative stress and chronic inflammation – important mechanisms of telomere attrition. Smoking is the most important lifestyle-associated risk factor for cardiovascular disease (Yusuf et al. 2004, Cao et al. 2013). In the past, LTL shortening had been associated with history of smoking in a cross-sectional design (Valdes et al. 2005). Recently, these findings have been confirmed in longitudinal studies (Bendix et al. 2014). Smoking does have a negative effect on LTL. Diabetes mellitus is a well-established risk factor for CVD (Ishida et al. 2012, 2013). In a cross-sectional study, diabetes type 2 was associated with decreased LTL (Salpea et al. 2010). Longitudinal studies found the same association (Gardner et al. 2005, You et al. 2012, Zhao et al. 2014), but minor limitations remain. Whether LTL is associated with diabetes

Knockout of Density-Enhanced Phosphatase-1 Impairs Cerebrovascular Reserve Capacity in an Arteriogenesis Model in Mice

Collateral growth, arteriogenesis, represents a proliferative mechanism involving endothelial cells, smooth muscle cells, and monocytes/macrophages. Here we investigated the role of Density-Enhanced Phosphatase-1 (DEP-1) in arteriogenesis in vivo, a protein-tyrosine-phosphatase that has controversially been discussed with regard to vascular cell biology. Wild-type C57BL/6 mice subjected to permanent left common carotid artery occlusion (CCAO) developed a significant diameter increase in distinct arteries of the circle of Willis, especially in the anterior cerebral artery. Analyzing the impact of loss of DEP-1 function, induction of collateralization was quantified after CCAO and hindlimb femoral artery ligation comparing wild-type and DEP-1−/− mice. Both cerebral collateralization assessed by latex perfusion and peripheral vessel growth in the femoral artery determined by microsphere perfusion and micro-CT analysis were not altered in DEP-1−/− compared to wild-type mice. Cerebrovascular reserve capacity, however, was significantly impaired in DEP-1−/− mice. Cerebrovascular transcriptional analysis of proarteriogenic growth factors and receptors showed specifically reduced transcripts of PDGF-B. SiRNA knockdown of DEP-1 in endothelial cells in vitro also resulted in significant PDGF-B downregulation, providing further evidence for DEP-1 in PDGF-B gene regulation. In summary, our data support the notion of DEP-1 as positive functional regulator in vascular cerebral arteriogenesis, involving differential PDGF-B gene expression.