Ivo R. Buschmann

Quantitative real-time RT-PCR data analysis: current concepts and the novel “gene expression’s C T difference” formula

For quantification of gene-specific mRNA, quantitative real-time RT-PCR has become one of the most frequently used methods over the last few years. This article focuses on the issue of real-time PCR data analysis and its mathematical background, offering a general concept for efficient, fast and precise data analysis superior to the commonly used comparative CT (ΔΔCT) and the standard curve method, as it considers individual amplification efficiencies for every PCR. This concept is based on a novel formula for the calculation of relative gene expression ratios, termed GED (Gene Expression’s CT Difference) formula. Prerequisites for this formula, such as real-time PCR kinetics, the concept of PCR efficiency and its determination, are discussed. Additionally, this article offers some technical considerations and information on statistical analysis of real-time PCR data.

Stimulation of arteriogenesis; a new concept for the treatment of arterial occlusive disease

After birth two forms of vessel growth can be observed; angiogenesis and arteriogenesis. Angiogenesis refers to the formation of capillary networks. Arteriogenesis refers to the growth of preexistent collateral arterioles leading to formation of large conductance arteries that are well capable to compensate for the loss of function of occluded arteries. The process of arteriogenesis is initiated when shear stresses increase in the preexistent collateral pathways upon narrowing of a main artery. The increased shear stress leads to an upregulation of cell adhesion molecules for circulating monocytes, which accumulate subsequently around the proliferating arteries and provide the several required cytokines and growth factors. Several strategies are currently tested for their potential to stimulate the process of arteriogenesis. These strategies focus either at shear stress, at direct stimulation of endothelial and smooth muscle cell growth or at the monocytic pathway and promising results were obtained from experimental studies. However, some important questions remain to be answered before arteriogenesis can be brought from bench to bedside.

The pathophysiology of the collateral circulation (arteriogenesis).

Since the mid 1980s a new strategy is coming from bench to bedside termed angiogenesis. This process involves sprouting of capillaries and finally results in newly developed microvessels which belong to the capillary level. Importantly these newly formed capillary tubes lack vascular smooth muscle cells, they are not surrounded by mural cells and are fragile and prone to rupture. Therefore these networks remain susceptible to hypoxic regulation, fail to become remodelled and are unable to sustain proper circulation: they cannot adapt to changes in physiological demands of blood supply. Since atherosclerosis affects large conductance arteries, capillary sprouting from compromised vessels cannot provide an adequate supply of blood flow to the endangered tissue. However, the body provides a natural system of pre‐existing collateral arteries, which may bypass sites of arterial occlusion. These vessels can dramatically increase their lumen by growth so as to provide enhanced perfusion to the jeopardized ischaemic regions. This process – termed arteriogenesis – finally results in fully functional and structurally normal arteries which can ameliorate the ensuing detrimental effects of vessel obstruction in many regions of the body. Hallmarks of arteriogenesis are increased levels of shear forces (rather than ischaemia), the invasion of circulating monocytes (and their pluripotent precursors), and the substrates of arteriogenesis are pre‐existing colateral arterioles. Copyright

Time course of arteriogenesis following femoral artery occlusion in the rabbit.

OBJECTIVE We examined the time course of arteriogenesis (collateral artery growth) after femoral artery ligation and the effect of monocyte chemoattractant protein-1 (MCP-1). METHODS New Zealand White rabbits received MCP-1 or phosphate buffered saline (PBS) for a 1-week period, either directly or 3 weeks after femoral artery ligation (non-ischemic model). A control group was studied with intact femoral arteries and another 1 min after acute femoral artery ligation. RESULTS Collateral conductance index significantly increased when MCP-1 treatment started directly after femoral artery ligation (acute occlusion: 0.94+/-0.19; without occlusion: 168.56+/-15.99; PBS: 4.10+/-0.48; MCP-1: 33.96+/-1.76 ml/min/100 mmHg). However, delayed onset of treatment 3 weeks after ligation and final study of conductance at 4 weeks showed no significant difference against a 4-week control (PBS: 79.08+/-7.24; MCP-1: 90.03+/-8.73 ml/min/100 mmHg). In these groups increased conductance indices were accompanied by a decrease in the number of visible collateral vessels (from 18 to 36 identifiable vessels at day 7 to about four at 21 days). CONCLUSION We conclude that the chemokine MCP-1 markedly accelerated collateral artery growth but did not alter its final extent above that reached spontaneously as a function of time. We show thus for the first time that a narrow time window exists for the responsiveness to the arteriogenic actions of MCP-1, a feature that MCP-1 may share with other growth factors. We show furthermore that the spontaneous adaptation by arteriogenesis stops when only about 50% of the vasodilatory reserve of the arterial bed before occlusion are reached. The superiority of few large arterial collaterals in their ability to conduct large amounts of blood flow per unit of pressure as compared to the angiogenic response where large numbers of small vessels are produced with minimal ability to allow mass transport of bulk flow is stressed.

GM-CSF: a strong arteriogenic factor acting by amplification of monocyte function

We investigated the role of the colony stimulating factor for monocytes (GM-CSF) to test the hypothesis whether prolongation of the monocytes life cycle will support arteriogenesis (rapid growth of preexisting collateral arteries). This appeared logical in view of our discovery that circulating monocytes play an important part in the positive remodeling of small preexisting arterioles into arteries to compensate for arterial occlusions (arteriogenesis) and especially following our findings that MCP-1 markedly increases the speed of arteriogenesis. The continuous infusion of GM-CSF for 7 days into the proximal stump of the acutely occluded femoral artery of rabbits by osmotic minipump produced indeed a marked arteriogenic response as demonstrated by an increase (2-fold) in number and size of collateral arteries on postmortem angiograms and by the increase of maximal blood flow during vasodilation measured in vivo by blood pump perfusion of the hindquarter (5-fold). When GM-CSF and MCP-1 were simultaneously infused the effects on arteriogenesis were additive on angiograms as well as on conductance. GM-CSF was also able to widen the time window of MCP-1 activity: MCP-1 treatment alone was ineffective when given after the third week following occlusion. When administered together with GM-CSF about 80% of normal maximal conductance of the artery that was replaced by collaterals were achieved, a result that was not reached before by any other experimental treatment. Experiments with cells isolated from treated animals showed that monocyte apoptosis was markedly reduced. In addition we hypothesize that GM-CSF may aid in releasing pluripotent monocyte (stem-) cells from the bone marrow into the circulation. In contrast to MCP-1, GM-CSF showed no activity on monocyte transmigration through- and also no influence on monocyte adhesion to cultured endothelial cells. In conclusion we have discovered a new function of the hemopoietic stem cell factor GM-CSF, which is also a powerful arteriogenic peptide that acts via prolongation of the life cycle of monocytes/macrophages.

Arteriogenesis Proceeds via ICAM-1/Mac-1- Mediated Mechanisms

Monocyte adhesion to shear stress–activated endothelium stands as an important initial step during arteriogenesis (collateral artery growth). Using multiple approaches, we tested the hypothesis that monocyte adhesion via intercellular adhesion molecule-1 (ICAM-1) and selectin interactions is essential for adaptive arteriogenesis. Forty-eight New Zealand White rabbits received either solvent, monocyte chemoattractant protein-1 (MCP-1) alone, MCP-1 plus ICAM-mab, or MCP-1 plus an IgG2a isotype control via osmotic minipumps. After 7 days, collateral conductance was evaluated: solvent 4.01 (mL/min per 100 mm Hg), MCP-1 plus ICAM-mab 8.04 (versus solvent P =NS), and MCP-1 alone 33.11 (versus solvent P <0.05). Furthermore, the right femoral arteries of ICAM-1−/−, Mac-1−/− and mice having defective selectin interactions (FT4/7−/−) as well as their corresponding controls were ligated. One week later, perfusion ratios were determined by the use of fluorescent microspheres. FT4/7−/− mice did not show any significant difference in perfusion restoration whereas ICAM-1−/− and Mac-1−/− mice had a significant reduction in arteriogenesis as compared with matching controls (FT4/7-WT 37±9%, FT4/7−/− 32±3%, P =0.31; C57BL/6J 59±9%, ICAM-1−/− 36±8%, P <0.05; Mac-1−/− 42±3%, P <0.05). ICAM-1/Mac-1–mediated monocyte adhesion to the endothelium of collateral arteries is an essential step for arteriogenesis, whereas this process can proceed via selectin interaction independent mechanisms. Furthermore, in vivo treatment with monoclonal antibodies against ICAM-1 totally abolishes the stimulatory effect of MCP-1 on collateral artery growth, suggesting that the mechanism of the MCP-1–induced arteriogenesis proceeds via the localization of monocytes rather than the action of the MCP-1 molecule itself.

Exogenous application of transforming growth factor beta 1 stimulates arteriogenesis in the peripheral circulation

Increased expression of transforming growth factor β1 (TGF‐ß1) during collateral artery growth, as well as its numerous effects on monocytes/macrophages and the smooth muscle cell cycle and differentiation, suggest a modulating role for this growth factor during arteriogenesis. We studied the effects of exogenously applied TGF‐ß1 on arteriogenesis as well as its interactions with monocytes, endothelial cells, and smooth muscle cells. In a New Zealand White (NZW) rabbit model of femoral artery ligation, increased expression of active TGF‐ß1 was found around proliferating arteries in NZW rabbits. The exogenous application of TGF‐ß1 led to an increase in both the number of visible collateral arteries as well as the conductance of the collateral circulation (4.0 ± 0.5 ml/min/100 mmHg vs. 28.9 ± 3.7 ml/min/100 mmHg, P < 0.05). Fluorescence activated cell sorting analysis showed an increase in the expression of the MAC‐1 receptor in both rabbit and human monocytes after treatment with TGF‐ß1 (control: 91.2 ± 4.2/482 ± 21.7; TGF‐ß1 200 ng/ml 193.9 ± 6.7/ 675.5 ± 25.7, P < 0.05 for all differences). TGF‐ß1 treated monocytes showed an increased endothelial adhesion and transmigration in transendothelial migration assays (5.75 ± 0.63 × 105 vs. 10.11 ± 0.04 × 105, P < 0.05). TGF‐ß1 had no direct pro‐angiogenic effect on human umbilical vein endothelial cells in a spheroid model of angiogenesis and inhibited the angiogenic effects of vascular endothelial growth factor.

Direct Evidence for Tumor Necrosis Factor-α Signaling in Arteriogenesis

Background—Arteriogenesis serves as an efficient mechanism for flow restoration after arterial occlusion. This process is associated with inflammatory mediators such as tumor necrosis factor-&agr; (TNF-&agr;), although their role in arteriogenesis remains unclear. We hypothesized that arteriogenesis is reduced in mice lacking functional TNF-&agr; or p55 receptor. To test this hypothesis, we developed a novel microsphere-based murine model of hindlimb perfusion measurement. Methods and Results—Unilateral femoral arteries of nude (n=9), TNF-&agr;−/− (n=9), TNF-&agr; receptor p55−/− (n=8), and p75−/− (n=8) mice as well as their appropriate genetic background controls were occluded. The nude mice underwent laser Doppler hindlimb flux measurements preoperatively, postoperatively, and after 7 days. Seven days after ligation, all animals underwent tissue perfusion determinations using fluorescent microspheres. Laser Doppler findings confirmed acute decrease in flux with falsely normal values after 1 week. Microsphere results from control mice showed perfusion restoration to values ≈50% of normal within 7 days. TNF-&agr;−/− mice demonstrated a significant reduction (45.1%) in collateral artery perfusion compared with controls (TNF-&agr;−/− 22.4±5.1% versus B6x129 49.7±9.3%;P <0.01). p55−/− mice exhibited an almost identical 45.8% reduction in collateral artery formation (p55−/− 28.3±4.3% versus C57BL/6J 61.8±9.1%;P <0.01), whereas p75−/− mice were equivalent to controls (p75−/− 54.5±5.5%;P =0.13). Conclusions—Microsphere techniques in mice offer a tool for the molecular dissection of arteriogenesis mechanisms. These results suggest that TNF-&agr; positively modulates arteriogenesis probably via signaling through its p55 receptor.

START Trial: a pilot study on STimulation of ARTeriogenesis using subcutaneous application of granulocyte-macrophage colony-stimulating factor as a new treatment for peripheral vascular disease.

Background—Granulocyte-macrophage colony-stimulating factor (GM-CSF) was recently shown to increase collateral flow index in patients with coronary artery disease. Experimental models showed beneficial effects of GM-CSF on collateral artery growth in the peripheral circulation. Thus, in the present study, we evaluated the effects of GM-CSF in patients with peripheral artery disease. Methods and Results—A double-blinded, randomized, placebo-controlled study was performed in 40 patients with moderate or severe intermittent claudication. Patients were treated with placebo or subcutaneously applied GM-CSF (10 &mgr;g/kg) for a period of 14 days (total of 7 injections). GM-CSF treatment led to a strong increase in total white blood cell count and C-reactive protein. Monocyte fraction initially increased but thereafter decreased significantly as compared with baseline. Both the placebo group and the treatment group showed a significant increase in walking distance at day 14 (placebo: 127±67 versus 184±87 meters, P=0.03, GM-CSF: 126±66 versus 189±141 meters, P=0.04) and at day 90. Change in walking time, the primary end point of the study, was not different between groups. No change in ankle-brachial index was found on GM-CSF treatment at day 14 or at day 90. Laser Doppler flowmetry measurements showed a significant decrease in microcirculatory flow reserve in the control group (P=0.03) and no change in the GM-CSF group. Conclusions—The present study does not support the use of GM-CSF for treatment of patients with moderate or severe intermittent claudication. Issues that need to be addressed are dosing, the selection of patients, and potential differences between GM-CSF effects in the coronary and the peripheral circulation.

Therapeutic Induction of Arteriogenesis in Hypoperfused Rat Brain Via Granulocyte-Macrophage Colony-Stimulating Factor

Background—Colony-stimulating factors (CSFs) have been shown to effectively induce arteriogenesis in the hindlimb. Moreover, clinical trials demonstrated positive effects of CSFs on arteriogenesis in patients with coronary artery disease. However, patients with cerebrovascular disease have not yet profited from treatments aimed at the growth of brain vessels. Thus far, angiogenesis studies have failed to demonstrate improvement of stroke outcome. Arteriogenesis differs from angiogenesis in that it substitutes arterial collaterals for the occluded artery. Methods and Results—We tested in a novel brain arteriogenesis rat model (occlusion of vertebral plus left carotid artery [3-VO]) the application of CSFs or saline over 7 or 21 days. On 3-VO postmortem, latex perfusion demonstrated a time- and treatment-dependent arteriogenesis of the posterior cerebral artery (PCA). In saline-treated animals, the PCA diameter increased by 39%; in granulocyte-macrophage (GM)-CSF-treated animals, this increase was significantly faster (72% after 1 week). Functionally, saline-treated animals exhibited a decline of CO2 reactivity (mm Hg) from 1.48% to 0.1% compared with GM-CSF–treated animals (1.43% arterial pCo2 change after 1 week). This difference remained significant after 3 weeks. This functional improvement correlated with increased numbers of CD68-positive macrophages in histological sections of the PCA in GM-CSF–treated animals and only a few macrophages in saline-treated animals. Conclusions—To the best of our knowledge, this is the first report of stimulation of arteriogenesis in the brain. The subcutaneous application of GM-CSF led to functional improvement of brain hemodynamic parameters.