A technique to detect and to quantify fasciocutaneous blood vessels in small laboratory animals ex vivo

PURPOSE: A microangiographical technique is described, which allows visualization of small and capillary blood vessels and quantification of fasciocutaneous blood vessels by means of digital computer analysis in very small laboratory animals. MATERIALS AND METHODS: The left carotid artery of 20 nu/nu mice was cannulated (26 gauge) and a mixture of gelatin, bariumsulfate, and green ink was injected according to standardized protocol. Fasciocutaneous blood vessels were visualized by digital mammography and analyzed for vessel length and vessel surface area as standardized units [SU] by computer program. RESULTS: With the described microangiography method, fasciocutaneous blood vessels down to capillary size level can be clearly visualized. Regions of interest (ROIs) can be defined and the containing vascular network quantified. Comparable results may be obtained by calculating the microvascular area index (MAI) and the microvascular length index (MLI), related to the ROIs size. Identical ROIs showed a high reproducibility for measured [SU] < 0.01 +/- 0.0012%. CONCLUSION: Combining microsurgical techniques, pharmacological knowledge, and modern digital image technology, we were able to visualize small and capillary blood vessels even in small laboratory animals. By using our own computer analytical program, quantification of vessels was reliable, highly reproducible, and fast.

Functional interaction of vascular endothelial-protein-tyrosine phosphatase with the angiopoietin receptor Tie-2

During development of the vertebrate vascular system essential signals are transduced via protein-tyrosine phosphorylation. Null-mutations of receptor-tyrosine kinase (RTK) genes expressed in endothelial cells (ECs) display early lethal vascular phenotypes. We aimed to identify endothelial protein-tyrosine phosphatases (PTPs), which should have similar importance in EC-biology. A murine receptor-type PTP was identified by a degenerated PCR cloning approach from endothelial cells (VE-PTP). By in situ hybridization this phosphatase was found to be specifically expressed in vascular ECs throughout mouse development. In experiments using GST-fusion proteins, as well as in transient transfections, trapping mutants of VE-PTP co-precipitated with the Angiopoietin receptor Tie-2, but not with the Vascular Endothelial Growth Factor receptor 2 (VEGFR-2/Flk-1). In addition, VE-PTP dephosphorylates Tie-2 but not VEGFR-2. We conclude that VE-PTP is a Tie-2 specific phosphatase expressed in ECs, and VE-PTP phosphatase activity serves to specifically modulate Angiopoietin/Tie-2 function. Based on its potential role as a regulator of blood vessel morphogenesis and maintainance, VE-PTP is a candidate gene for inherited vascular malformations similar to the Tie-2 gene.

[Calcium and blood pressure]

The regulation of blood pressure is complex with several organs being involved. Intracellular calcium plays a crucial role in the regulation of cardiovascular functions: An increased influx of calcium into the vascular smooth muscle cells leads to an augmental muscular tone and therefore to an increased vascular resistance and rise in blood pressure. Parathormone plays a permissive role since it regulates the calcium-influx into the cells and thus increases the vasoconstrictive effect. There is a positive correlation between parathormone and blood pressure, present in primary as well as secondary hyperparathyroidism. Moreover, patients with essential hypertension have high parathormone levels already before hypertension is diagnosed. A calcium-rich diet (> 1000 mg calcium daily) slightly decreases blood pressure. This positive effect is due to parathormone suppression with a subsequently decreased calcium content in the vascular smooth muscle cells. A calcium-rich diet inhibits lipogenesis in the fat tissue; thus additionally improving the cardiovascular risk profile.

Methylprednisolone Fails to Preserve Pulmonary Surfactant and Blood-Air Barrier Integrity in a Porcine Cardiopulmonary Bypass Model

BACKGROUND: Pulmonary inflammation after cardiac surgery with cardiopulmonary bypass (CPB) has been linked to respiratory dysfunction and ultrastructural injury. Whether pretreatment with methylprednisolone (MP) can preserve pulmonary surfactant and blood-air barrier, thereby improving pulmonary function, was tested in a porcine CPB-model. MATERIALS AND METHODS: After randomizing pigs to placebo (PLA; n = 5) or MP (30 mg/kg, MP; n = 5), animals were subjected to 3 h of CPB with 1 h of cardioplegic cardiac arrest. Hemodynamic data, plasma tumor necrosis factor-alpha (TNF-alpha, ELISA), and pulmonary function parameters were assessed before, 15 min after CPB, and 8 h after CPB. Lung biopsies were analyzed for TNF-alpha (Western blot) or blood-air barrier and surfactant morphology (electron microscopy, stereology). RESULTS: Systemic TNF-alpha increased and cardiac index decreased at 8 h after CPB in PLA (P < 0.05 versus pre-CPB), but not in MP (P < 0.05 versus PLA). In both groups, at 8 h after CPB, PaO(2) and PaO(2)/FiO(2) were decreased and arterio-alveolar oxygen difference and pulmonary vascular resistance were increased (P < 0.05 versus baseline). Postoperative pulmonary TNF-alpha remained unchanged in both groups, but tended to be higher in PLA (P = 0.06 versus MP). The volume fraction of inactivated intra-alveolar surfactant was increased in PLA (58 +/- 17% versus 83 +/- 6%) and MP (55 +/- 18% versus 80 +/- 17%) after CPB (P < 0.05 versus baseline for both groups). Profound blood-air barrier injury was present in both groups at 8 h as indicated by an increased blood-air barrier integrity score (PLA: 1.28 +/- 0.03 versus 1.70 +/- 0.1; MP: 1.27 +/- 0.08 versus 1.81 +/- 0.1; P < 0.05). CONCLUSION: Despite reduction of the systemic inflammatory response and pulmonary TNF-alpha generation, methylprednisolone fails to decrease pulmonary TNF-alpha and to preserve pulmonary surfactant morphology, blood-air barrier integrity, and pulmonary function after CPB.

Distinct roles of vascular endothelial growth factor-D in lymphangiogenesis and metastasis

In many human carcinomas, expression of the lymphangiogenic factor vascular endothelial growth factor-D (VEGF-D) correlates with up-regulated lymphangiogenesis and regional lymph node metastasis. Here, we have used the Rip1Tag2 transgenic mouse model of pancreatic beta-cell carcinogenesis to investigate the functional role of VEGF-D in the induction of lymphangiogenesis and tumor progression. Expression of VEGF-D in beta cells of single-transgenic Rip1VEGF-D mice resulted in the formation of peri-insular lymphatic lacunae, often containing leukocyte accumulations and blood hemorrhages. When these mice were crossed to Rip1Tag2 mice, VEGF-D-expressing tumors also exhibited peritumoral lymphangiogenesis with lymphocyte accumulations and hemorrhages, and they frequently developed lymph node and lung metastases. Notably, tumor outgrowth and blood microvessel density were significantly reduced in VEGF-D-expressing tumors. Our results demonstrate that VEGF-D induces lymphangiogenesis, promotes metastasis to lymph nodes and lungs, and yet represses hemangiogenesis and tumor outgrowth. Because a comparable transgenic expression of vascular endothelial growth factor-C (VEGF-C) in Rip1Tag2 has been shown previously to provoke lymphangiogenesis and lymph node metastasis in the absence of any distant metastasis, leukocyte infiltration, or angiogenesis-suppressing effects, these results reveal further functional differences between VEGF-D and VEGF-C.

Placental growth factor-1 attenuates vascular endothelial growth factor-A-dependent tumor angiogenesis during beta cell carcinogenesis

Members of the vascular endothelial growth factor (VEGF) family are critical players in angiogenesis and lymphangiogenesis. Although VEGF-A has been shown to exert fundamental functions in physiologic and pathologic angiogenesis, the exact role of the VEGF family member placental growth factor (PlGF) in tumor angiogenesis has remained controversial. To gain insight into PlGF function during tumor angiogenesis, we have generated transgenic mouse lines expressing human PlGF-1 in the beta cells of the pancreatic islets of Langerhans (Rip1PlGF-1). In single-transgenic Rip1PlGF-1 mice, intra-insular blood vessels are found highly dilated, whereas islet physiology is unaffected. Upon crossing of these mice with the Rip1Tag2 transgenic mouse model of pancreatic beta cell carcinogenesis, tumors of double-transgenic Rip1Tag2;Rip1PlGF-1 mice display reduced growth due to attenuated tumor angiogenesis. The coexpression of transgenic PlGF-1 and endogenous VEGF-A in the beta tumor cells of double-transgenic animals causes the formation of low-angiogenic hPlGF-1/mVEGF-A heterodimers at the expense of highly angiogenic mVEGF-A homodimers resulting in diminished tumor angiogenesis and reduced tumor infiltration by neutrophils, known to contribute to the angiogenic switch in Rip1Tag2 mice. The results indicate that the ratio between the expression levels of two members of the VEGF family of angiogenic factors, PlGF-1 and VEGF-A, determines the overall angiogenic activity and, thus, the extent of tumor angiogenesis and tumor growth.

Acute device-based blood pressure reduction: electrical activation of the carotid baroreflex in patients undergoing elective carotid surgery

Carotid sinus baroreceptors are involved in controlling blood pressure (BP) by providing input to the cardiovascular regulatory centers of the medulla. The acute effect of temporarily placing an electrode on the carotid sinus wall to electrically activate the baroreflex was investigated. We studied 11 patients undergoing elective carotid surgery. Baseline BP was 146+30/66+/-17 mm Hg and heart rate (HR) 72+/-7 bpm (mean +/- standard deviation). An electrode was placed upon the carotid sinus and after obtaining a steady state baseline of BP and HR, an electric current was applied and increased in 1-volt increments. A voltage dependent and highly significant reduction in BP was observed which averaged 18+/-26* and 8.0+/-12 mm Hg for systolic BP and diastolic BP, respectively. Maximal reductions occurred at 4.4+/-1.2 V: 23+/-24 mm Hg*, 16+/-10 mm Hg* and 7+/-12 bpm* for systolic BP, diastolic BP and HR, respectively ( = p <.05). Thus, electrical stimulation of the carotid sinus activates the carotid baroreflex resulting in a reduction in BP and HR. This presents a proof of concept for device based baroreflex modulation in acute BP regulation and adds to the available data which provide a rationale for evaluating this system in the context of chronic BP reduction in hypertensive patients.

Effects of an aging vascular model on healthy and diseased hearts

The vitamin D(3) and nicotine (VDN) model is a model of isolated systolic hypertension (ISH) due to arterial calcification raising arterial stiffness and vascular impedance similar to an aged and stiffened arterial tree. We therefore analyzed the impact of this aging model on normal and diseased hearts with myocardial infarction (MI). Wistar rats were treated with VDN (n = 9), subjected to MI by coronary ligation (n = 10), or subjected to a combination of both MI and VDN treatment (VDN/MI, n = 14). A sham-treated group served as control (Ctrl, n = 10). Transthoracic echocardiography was performed every 2 wk, whereas invasive indexes were obtained at week 8 before death. Calcium, collagen, and protein contents were measured in the heart and the aorta. Systolic blood pressure, pulse pressure, thoracic aortic calcium, and end-systolic elastance as an index of myocardial contractility were highest in the aging model group compared with MI and Ctrl groups (P(VDN) < 0.05, 2-way ANOVA). Left ventricular wall stress and brain natriuretic peptide (P(VDNxMI) = not significant) were highest, while ejection fraction, stroke volume, and cardiac output were lowest in the combined group versus all other groups (P(VDNxMI) < 0.05). The combination of ISH due to this aging model and MI demonstrates significant alterations in cardiac function. This model mimics several clinical phenomena of cardiovascular aging and may thus serve to further study novel therapies.

Transient raise of endothelin-1 plasma level and reduction of ocular blood flow in a patient with optic neuritis

PURPOSE: To analyze how far an ischemic component might have been involved in optic neuritis. METHODS: Case report: a 32-year-old man with symptoms characteristic for optic neuritis underwent extensive clinical, laboratory/serological and vascular examination for systemic associations and vascular involvement. RESULTS: The patient was found to have a temporary ocular blood flow dysregulation and increased plasma endothelin-1 levels which decreased after the acute phase of the optic nerve. CONCLUSIONS: We conclude that there might be an ischemic component in this patient with optic neuritis and hypothesize that this ischemic component is at least in part due to a temporarily increased endothelin-1 level.

Vascular dysfunction and reduced circulating endothelial progenitor cells in young healthy UK South Asian men

OBJECTIVES: The objective of this study was to examine determinants of excess coronary artery disease risk in UK South Asians, more prevalent in this population than UK Caucasians, by examining differences in risk factors, vascular function, and endothelial progenitor cells (EPCs). METHODS AND RESULTS: 24 South Asian and 25 Caucasian healthy age-matched nonsmoking men were studied. Vascular function was assessed by flow-mediated and GTN brachial artery dilatation and blood flow responses to infusion of ACh, SNP, and L-NMMA. EPC number and function were measured by flow cytometry (CD34, CD133, and KDR positive cells), and CFU/migration assays. Traditional risk factors and anthropometric measurements were similar in the groups. South Asians had higher fasting insulin levels (6.01 versus 3.62 microU/mL; P = 0.02). South Asians had lower FMD (6.9 versus 8.5%; P = 0.003), L-NMMA response (0.8 versus 1.3 mL/min/100 mL; P = 0.03), mean SNP response (9.5+/-0.6 versus 11.6+/-0.6; P = 0.02), EPC number (0.046+/-0.005% versus 0.085+/-0.009%; P = < 0.001), and CFU ability (CFU 4.29+/-1.57 versus 18.86+/-4.00; P = 0.005). EPC number was the strongest predictor of FMD. Ethnicity was the strongest predictor of EPC number. CONCLUSIONS: Healthy South Asian men are more insulin resistant, and demonstrate endothelial dysfunction and reduced EPC number and function compared with Caucasians. These abnormalities may contribute to their increased CAD risk.

The immediate and sustained effects of volume challenge on regional blood flows in pigs

BACKGROUND: The postoperative assessment of volume status is not straightforward because of concomitant changes in intravascular volume and vascular tone. Hypovolemia and blood flow redistribution may compromise the perfusion of the intraabdominal organs. We investigated the effects of a volume challenge in different intra- and extraabdominal vascular beds. METHODS: Twelve pigs were studied 6 h after major intraabdominal surgery under general anesthesia when clinically normovolemic. Volume challenges consisted of 200 mL rapidly infused 6% hydroxyethyl starch. Systemic (continuous thermodilution) and regional (ultrasound Doppler) flows in carotid, renal, celiac trunk, hepatic, and superior mesenteric arteries and the portal vein were continuously measured. The acute and sustained effects of the challenge were compared with baseline. RESULTS: Volume challenge produced a sustained increase of 22% +/- 15% in cardiac output (P < 0.001). Blood flow increased by 10% +/- 9% in the renal artery, by 22% +/- 15% in the carotid artery, by 26% +/- 15% in the superior mesenteric artery, and by 31% +/- 20% in the portal vein (all P < 0.001). Blood flow increases in the celiac trunk (8% +/- 13%) and the hepatic artery (7% +/- 19%) were not significant. Increases in regional blood flow occurred early and were sustained. Mean arterial and central venous blood pressures increased early and decreased later (all P < 0.05). CONCLUSIONS: A volume challenge in clinically euvolemic postoperative animals was associated with a sustained increase in blood flow to all vascular beds, although the increase in the celiac trunk and the hepatic artery was very modest and did not reach statistical significance. Whether improved postoperative organ perfusion is accompanied by a lower complication rate should be evaluated in further studies.

Quantitative stress echocardiography in coronary artery disease using contrast-based myocardial blood flow measurements: prospective comparison with coronary angiography

AIM: To test whether quantitative stress echocardiography using contrast-based myocardial blood flow (MBF, ml x min(-1) x g(-1)) measurements can detect coronary artery disease in humans. METHODS: 48 patients eligible for pharmacological stress testing by myocardial contrast echocardiography (MCE) and willing to undergo subsequent coronary angiography were prospectively enrolled in the study. Baseline and adenosine-induced (140 microg x kg(-1) x min(-1)) hyperaemic MBF was analysed according to a three-coronary-artery-territory model. Vascular territories were categorised into three groups with increasing stenosis severity defined as percentage diameter reduction by quantitative coronary angiography. RESULTS: Myocardial blood flow reserve (MBFR)-that is, the ratio of hyperaemic to baseline MBF, was obtained in 128 (89%) territories. Mean (SD) baseline MBF was 1.073 (0.395) ml x min(-1) x g(-1) and did not differ between territories supplied by coronary arteries with mild (<50% stenosis), moderate (50%-74% stenosis) or severe (>or=75% stenosis) disease. Mean (SD) hyperaemic MBF and MBFR were 2.509 (1.078) ml x min(-1) x g(-1) and 2.54 (1.03), respectively, and decreased linearly (r2 = 0.21 and r2 = 0.39) with stenosis severity. ROC analysis revealed that a territorial MBFR <1.94 detected >or=50% stenosis with 89% sensitivity and 92% specificity. CONCLUSION: Quantitative stress testing based on MBF measurements derived from contrast echocardiography is a new method for the non-invasive and reliable assessment of coronary artery disease in humans.

Angiotensinergic neurons in sympathetic coeliac ganglia innervating rat and human mesenteric resistance blood vessels

In contrast to the current belief that angiotensin II (Ang II) interacts with the sympathetic nervous system only as a circulating hormone, we document here the existence of endogenous Ang II in the neurons of rat and human sympathetic coeliac ganglia and their angiotensinergic innervation with mesenteric resistance blood vessels. Angiotensinogen - and angiotensin converting enzyme-mRNA were detected by using quantitative real time polymerase chain reaction in total RNA extracts of rat coeliac ganglia, while renin mRNA was untraceable. Cathepsin D, a protease responsible for cleavage beneath other substrates also angiotensinogen to angiotensin I, was successfully detected in rat coeliac ganglia indicating the possibility of existence of alternative pathways. Angiotensinogen mRNA was also detected by in situ hybridization in the cytoplasm of neurons of rat coeliac ganglia. Immunoreactivity for Ang II was demonstrated in rat and human coeliac ganglia as well as with mesenteric resistance blood vessels. By using confocal laser scanning microscopy we were able to demonstrate the presence of angiotensinergic synapses en passant along side of vascular smooth muscle cells. Our findings indicate that Ang II is synthesized inside the neurons of sympathetic coeliac ganglia and may act as an endogenous neurotransmitter locally with the mesenteric resistance blood vessels.

The role of purinergic signaling in the liver and in transplantation: effects of extracellular nucleotides on hepatic graft vascular injury, rejection and metabolism

Extracellular nucleotides (e.g. ATP, UTP, ADP) are released by activated endothelium, leukocytes and platelets within the injured vasculature and bind specific cell-surface type-2 purinergic (P2) receptors. This process drives vascular inflammation and thrombosis within grafted organs. Importantly, there are also vascular ectonucleotidases i.e. ectoenzymes that hydrolyze extracellular nucleotides in the blood to generate nucleosides (viz. adenosine). Endothelial cell NTPDase1/CD39 has been shown to critically modulate levels of circulating nucleotides. This process tends to limit the activation of platelet and leukocyte expressed P2 receptors and also generates adenosine to reverse inflammatory events. This vascular protective CD39 activity is rapidly inhibited by oxidative reactions, such as is observed with liver ischemia reperfusion injury. In this review, we chiefly address the impact of these signaling cascades following liver transplantation. Interestingly, the hepatic vasculature, hepatocytes and all non-parenchymal cell types express several components co-ordinating the purinergic signaling response. With hepatic and vascular dysfunction, we note heightened P2- expression and alterations in ectonucleotidase expression and function that may predispose to progression of disease. In addition to documented impacts upon the vasculature during engraftment, extracellular nucleotides also have direct influences upon liver function and bile flow (both under physiological and pathological states). We have recently shown that alterations in purinergic signaling mediated by altered CD39 expression have major impacts upon hepatic metabolism, repair mechanisms, regeneration and associated immune responses. Future clinical applications in transplantation might involve new therapeutic modalities using soluble recombinant forms of CD39, altering expression of this ectonucleotidase by drugs and/or using small molecules to inhibit deleterious P2-mediated signaling while augmenting beneficial adenosine-mediated effects within the transplanted liver.

IL-6 transsignalling modulates the early effector phase of EAE and targets the blood-brain barrier

Interleukin-6 (IL-6) plays a crucial role in the pathogenesis of experimental autoimmune encephalomyelitis (EAE). It exerts its cellular effects by a membrane-bound IL-6 receptor (IL-6R), or, alternatively, by forming a complex with the soluble IL-6R (sIL-6R), a process named IL-6 transsignalling. Here we investigate the role of IL-6 transsignalling in myelin basic protein (MBP)-induced EAE in the Lewis rat. In vivo blockade of IL-6 transsignalling by the injection of a specifically designed gp130-Fc fusion protein significantly delayed the onset of adoptively transferred EAE in comparison to control rats injected with PBS or isotype IgG. Histological evaluation on day 3 after immunization revealed reduced numbers of T cells and macrophages in the lumbar spinal cord of gp130-Fc treated rats. At the same time, blockade of IL-6 transsignalling resulted in a reduced expression of vascular cell adhesion molecule-1 on spinal cord microvessels while experiments in cell culture failed to show a direct effect on the regulation of endothelial adhesion molecules. In experiments including active EAE and T cell culture, inhibition of IL-6 transsignalling mildly increased T cell proliferation, but did not change severity of active MBP-EAE or regulate Th1/Th17 responses. We conclude that IL-6 transsignalling may play a role in autoimmune inflammation of the CNS mainly by regulating early expression of adhesion molecules, possibly via cellular networks at the blood-brain barrier.