Cerebral arteriogenesis is enhanced by pharmacological as well as fluid-shear-stress activation of the Trpv4 calcium channel.

OBJECTIVES: This study aimed to determine the importance of the shear-stress-sensitive calcium channels Trpc1, Trpm7, Trpp2, Trpv2 (transient receptor potential cation channel, subfamily V, member 2) and Trpv4 for cerebral arteriogenesis. The expression profiles were analysed, comparing the stimulation of collateral growth by target-specific drugs to that achieved by maximum increased fluid shear stress (FSS). DESIGN: A prospective, controlled study wherein rats were subjected to bilateral carotid artery ligature (BCL), or BCL + arteriovenous fistula, or BCL + drug application. METHODS: Messenger RNA (mRNA) abundance and protein expression were determined in FSS-stimulated cerebral collaterals by quantitative real-time polymerase chain reaction (qRT-PCR) and immunohistochemistry. Drugs were applied via osmotic mini pumps and arteriogenesis was evaluated by post-mortem angiograms and Ki67 immunostaining. RESULTS: Trpv4 was the only mechanosensitive Trp channel showing significantly increased mRNA abundance and protein expression after FSS stimulation. Activation of Trpv4 by 4α-phorbol-12,13-didecanoate caused significantly enhanced collateral growth (length: 4.43 ± 0.20 mm and diameter: 282.6 ± 8.1 µm) compared with control (length: 3.80 ± 0.06 mm and diameter: 237.3 ± 5.3 µm). Drug application stimulated arteriogenesis to almost the same extent as did maximum FSS stimulation (length: 4.61 ± 0.07 mm and diameter: 327.4 ± 12.6 µm). CONCLUSIONS: Trpv4 showed significantly increased expression in FSS-stimulated cerebral collaterals. Pharmacological Trpv4 activation enhanced cerebral arteriogenesis, pinpointing Trpv4 as a possible candidate for the development of new therapeutic concepts.

Therapeutic arteriogenesis in peripheral arterial disease: combining intervention and passive training.

The prevalence of peripheral arterial disease (PAD) is on the rise in an aging population, significantly affecting quality of life, morbidity and mortality. Besides medical treatment and surgical or interventional revascularization, supervised exercise programs are a primary treatment modality for PAD. Training may significantly increase pain-free walking time (+ 180 %) while avoiding the associated complications of (repeated) invasive revascularization. Training effects rely on an improvement of risk factor management, muscle function, economy of movement, hemorheology, vascular growth and collateral vessel growth. Exercise training upregulates pulsatile fluid shear stress on the vascular endothelium, prompting an improvement of endothelial function (eNOS, NO) and an outgrowth of preexistent collaterals (arteriogenesis) to functional conductance arteries outside the ischemic area at risk. However, the necessary intense minimum training intervals compromise patient compliance, and the impaired functional status of many PAD patients limits active exercise training. Strategies are necessary to a) increase training compliance, b) make the benefits of exercise training available to patients unable to exercise actively and c) therapeutically enhance the adaptive growth of biological bypasses (arteriogenesis). A modified form of “passive exercise training” derived from enhanced external counterpulsation (low-pressure ECP) which was originally developed for the therapy of heart failure, may prove to be an option for this group of patients. Therefore, this review article suggests a tailored combination therapy, consisting of a facilitating revascularization procedure to restore arterial inflow, succeeded by supervised exercise training, which has yielded promising therapeutic results in clinical trials. Further studies, using appropriate imaging methods and controls, are under way to (a) establish the efficacy of low-pressure EECP in PAD patients and (b) to directly correlate training-induced improvements of collateral flow to the functional improvements seen with exercise training.

Calcium-dependent signalling is essential during collateral growth in the pig hind limb-ischemia model.

We investigated the effect of pharmacological activation of the Ca(2+)-channel transient receptor potential cation channel, subfamily V, member 4 (TRPV4) on collateral growth in a pig hind limb-ischemia model thereby identifying subcellular mechanisms. Domestic pigs received femoral artery ligature and were randomly assigned to one of the following groups (each n=6): (1) 4alpha-phorbol 12,13-didecanoate (4alphaPDD) treatment; (2) treatment with an arterio-venous shunt (AV-shunt) distal to the occlusion; or (3) implantation of NaCl-filled minipump. Six sham-operated pigs acted as controls. Aortic and peripheral mean arterial pressure (MAP) measurements were performed to assess the collateral flow index (CFI). Tissue was isolated from M. quadriceps for immunohistochemistry and from isolated collateral arteries for quantitative real time PCR (qRT-PCR). Shortly after ligature the CFI dropped from 0.96+/-0.02 to 0.21+/-0.02 in all ligature-treated groups. In ligature-only-treated pigs CFI increased to 0.56+/-0.03 after 7days. Treatment with 4alphaPDD led to an enhancement of CFI compared with ligature alone (0.73+/-0.03). CD31-staining showed improved arteriolar density. Increased Ki67 staining in collaterals indicated proliferation. qRT-PCR and Western blot analysis showed upregulation or modulation of Ca(2+)-dependent transcription factors nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 1 (NFATc1), Kv channel interacting protein 3, calsenilin (KCNIP3/CSEN/DREAM), and myocyte enhancer factor 2C (MEF2C) in 4alphaPDD- and AV-shunt-treated pigs compared with controls. Improved CFI after 4alphaPDD treatment identifies TRPV4 as an initial fluid shear-stress sensor and collateral remodelling and growth trigger. Subcellularly, modulation of Ca(2+)-dependent transcription factors indicates a pivotal role for Ca(2+)-signalling during arteriogenesis.

Exercise linked to transient increase in expression and activity of cation channels in newly formed hind-limb collaterals.

OBJECTIVE: This study aimed to compare arteriogenesis after femoral artery occlusion as influenced by exercise or arteriovenous shunt and follow changes in collateral transient receptor potential cation channel, subfamily V, member 4 (Trpv4). DESIGN: A prospective, controlled study wherein rats were subjected to femoral artery ligation (FAL), or FAL+arteriovenous shunt. Collateral Trpv4 was determined 0.5 and 6h post exercise. METHODS: Rats were subjected to exercise for 15 min, twice daily. The number and diameter of collaterals were assessed after 7 days. Collateral Trpv4 expression was quantified by reverse transcription-polymerase chain reaction. RESULTS: Collateral number and diameter per limb were significantly higher in the shunt group (number: 16.0+/-2.4 and diameter: 216.0+/-34 microm) compared to the ligature (number: 9.4+/-2 and diameter: 144+/-21 microm) and exercise groups (number: 9.9+/-2.5 and diameter: 151+/-15 microm). Trpv4 expression in collaterals harvested 0.5h post exercise was not significantly different from expression in shunted rats. It was significantly lower in collaterals harvested 6h post exercise (comparable to that in ligated rats). CONCLUSION: Collateral formation was greater in the shunt group than in the exercise group. Exercise-induced Trpv4 up-regulation, not significantly different from that achieved with shunt, returned to control values when evaluated 6h post exercise. More frequent exercise to chronically increase fluid shear stress, as with a shunt model, may be required for sufficient arteriogenesis to compensate for peripheral occlusion.

Classically and alternatively activated macrophages contribute to tissue remodelling after myocardial infarction.

An important goal in cardiology is to minimize myocardial necrosis and to support a discrete but resilient scar formation after myocardial infarction (MI). Macrophages are a type of cells that influence cardiac remodelling during MI. Therefore, the goal of the present study was to investigate their transcriptional profile and to identify the type of activation during scar tissue formation. Ligature of the left anterior descending coronary artery was performed in mice. Macrophages were isolated from infarcted tissue using magnetic cell sorting after 5 days. The total RNA of macrophages was subjected to microarray analysis and compared with RNA from MI and LV-control. mRNA abundance of relevant targets was validated by quantitative real-time PCR 2, 5 and 10 days after MI (qRT-PCR). Immunohistochemistry was performed to localize activation type-specific proteins. The genome scan revealed 68 targets predominantly expressed by macrophages after MI. Among these targets, an increased mRNA abundance of genes, involved in both the classically (tumour necrosis factor alpha, interleukin 6, interleukin 1beta) and the alternatively (arginase 1 and 2, mannose receptor C type 1, chitinase 3-like 3) activated phenotype of macrophages, was found 5 days after MI. This observation was confirmed by qRT-PCR. Using immunohistochemistry, we confirmed that tumour necrosis factor alpha, representing the classical activation, is strongly transcribed early after ligature (2 days). It was decreased after 5 and 10 days. Five days after MI, we found a fundamental change towards alternative activation of macrophages with up-regulation of arginase 1. Our results demonstrate that macrophages are differentially activated during different phases of scar tissue formation after MI. During the early inflammatory phase, macrophages are predominantly classically activated, whereas their phenotype changes during the important transition from inflammation to scar tissue formation into an alternatively activated type.

The temporal and spatial distribution of macrophage subpopulations during arteriogenesis.

Chronic arterial occlusion leads to growth of collaterals - a process termed arteriogenesis, in which macrophages play a prominent role in remodelling and growth. However, a detailed analysis which of distinct macrophage subpopulations involved in arteriogenesis has never been performed. In the present study the temporal and spatial distribution of macrophage subtypes during arteriogenesis in a rat model with chronically elevated fluid shear stress (FSS) is investigated. Local macrophage subpopulations were histologically immuno-phenotyped using CD68 (a ubiquitous macrophage marker) and CD163, a specific M2 macrophage marker. Without occlusion few M2-macrophages reside in the perivascular space. Early after occlusion (12h) the number of M2 macrophages increases strongly and M1 macrophages begin emerging into the collateral. After 3 days they appear in the perivascular space. Both macrophage subtypes increase until 28d after treatment, whereas M2 macrophages dominate at the site of collateral growth. The local distribution of the subpopulations changes during the arteriogenic process. Whereas M1 macrophages are detected directly adjacent to the media, M2 macrophages are present in the most outer perivascular region of the growing collateral vessel. Systemic alterations of blood leucocytes in mice after femoral artery ligature (FAL) were investigated by FACS analysis of serial blood samples. During collateral remodelling histological changes were not reflected in circulating monocytes in the peripheral blood. The activation state of macrophages in mice with FAL was modulated by injections of either dexamethasone or the interleukins IL10 or IL3/IL14. The arteriogenic response was assessed by hind limb perfusion with laser Doppler measurements after 3, 7 and 14d. Suppressing inflammatory monocyte subtypes (M1) with dexamethasone led to impaired perfusion recovery after FAL in mice, whereas IL10 or IL4/IL13 application significantly increased perfusion recovery. This investigation demonstrates that a forced shift towards M2 macrophages improves the arteriogenic response. The distinct early increase and spatial distribution of M2 macrophages support the idea that this subtype plays a predominant role during collateral remodelling.