Isoproterenol Acts as a Biased Agonist of the Alpha-1A-Adrenoceptor that Selectively Activates the MAPK/ERK Pathway

The α_1A-AR is thought to couple predominantly to the Gα_q/PLC pathway and lead to phosphoinositide hydrolysis and calcium mobilization, although certain agonists acting at this receptor have been reported to trigger activation of arachidonic acid formation and MAPK pathways. For several G protein-coupled receptors (GPCRs) agonists can manifest a bias for activation of particular effector signaling output, i.e. not all agonists of a given GPCR generate responses through utilization of the same signaling cascade(s). Previous work with Gα_q coupling-defective variants of α_1A-AR, as well as a combination of Ca²⁺ channel blockers, uncovered cross-talk between α_1A-AR and β₂-AR that leads to potentiation of a Gα_q-independent signaling cascade in response to α_1A-AR activation. We hypothesized that molecules exist that act as biased agonists to selectively activate this pathway. In this report, isoproterenol (Iso), typically viewed as β-AR-selective agonist, was examined with respect to activation of α_1A-AR. α_1A-AR selective antagonists were used to specifically block Iso evoked signaling in different cellular backgrounds and confirm its action at α_1A-AR. Iso induced signaling at α_1A-AR was further interrogated by probing steps along the Gα_q /PLC, Gα_s and MAPK/ERK pathways. In HEK-293/EBNA cells transiently transduced with α_1A-AR, and CHO_α_1A-AR stable cells, Iso evoked low potency ERK activity as well as Ca²⁺ mobilization that could be blocked by α_1A-AR selective antagonists. The kinetics of Iso induced Ca²⁺ transients differed from typical Gα_q- mediated Ca²⁺ mobilization, lacking both the fast IP₃R mediated response and the sustained phase of Ca²⁺ re-entry. Moreover, no inositol phosphate (IP) accumulation could be detected in either cell line after stimulation with Iso, but activation was accompanied by receptor internalization. Data are presented that indicate that Iso represents a novel type of α_1A-AR partial agonist with signaling bias toward MAPK/ERK signaling cascade that is likely independent of coupling to Gα_q.     

Substance P and Chronic Pain in Patients with Chronic Inflammation of Connective Tissue

Objective

Evidence suggests that substance P (SP) is involved in chronic joint inflammation, such as the pathogenesis of rheumatoid arthritis and osteoarthritis. The goal of the research was to evaluate the correlation between chronic pain and changes in the SP level in patients with chronic inflammation of the connective tissue.

Methods

Patients with osteoarthritis and rheumatoid arthritis were enrolled in this study. The relationship between chronic pain intensity and the serum SP concentration was evaluated in these groups of patients with osteoarthritis and rheumatoid arthritis.

Results

The results showed a positive correlation between the serum SP concentrations and chronic pain intensity.

Conclusions

1. The SP serum concentration was significantly different between the groups of patients with OA and RA. 2. There was a positive correlation between the serum SP concentration and chronic pain intensity in OA and RA patients.     

Erythrocyte-associated transients in capillary PO2: an isovolemic hemodilution study in the rat spinotrapezius muscle

Mathematical simulations of oxygen delivery to tissue from capillaries that take into account the particulate nature of blood flow predict the existence of oxygen tension (Po(2)) gradients between erythrocytes (RBCs). As RBCs and plasma alternately pass an observation point, these gradients are manifested as rapid fluctuations in Po(2), also known as erythrocyte-associated transients (EATs). The impact of hemodilution on EATs and oxygen delivery at the capillary level of the microcirculation has yet to be elucidated. Therefore, in the present study, phosphorescence quenching microscopy was used to measure EATs and Po(2) in capillaries of the rat spinotrapezius muscle at the following systemic hematocrits (Hct(sys)): normal (39%) and after moderate (HES1; 27%) or severe (HES2; 15%) isovolemic hemodilution using a 6% hetastarch solution. A 532-nm laser, generating 10-micros pulses concentrated onto a 0.9-microm spot, was used to obtain plasma Po(2) values 100 times/s at points along surface capillaries of the muscle. Mean capillary Po(2) (Pc(O(2)); means +/- SE) significantly decreased between conditions (normal: 56 +/- 2 mmHg, n = 45; HES1: 47 +/- 2 mmHg, n = 62; HES2: 27 +/- 2 mmHg, n = 52, where n = capillary number). In addition, the magnitude of Po(2) transients (DeltaPo(2)) significantly decreased with hemodilution (normal: 19 +/- 1 mmHg, n = 45; HES1: 11 +/- 1 mmHg, n = 62; HES2: 6 +/- 1 mmHg, n = 52). Results suggest that the decrease in Pc(O(2)) and DeltaPo(2) with hemodilution is primarily dependent on Hct(sys) and subsequent microvascular compensations.     

Contributions of collision rate and collision efficiency to erythrocyte aggregation in postcapillary venules at low flow rates

Red blood cell aggregation at low flow rates increases venous vascular resistance, but the process of aggregate formation in these vessels is not well understood. We previously reported that aggregate formation in postcapillary venules of the rat spinotrapezius muscle mainly occurs in a middle region between 15 and 30 microm downstream from the entrance. In light of the findings in that study, the main purpose of this study was to test two hypotheses by measuring collision frequency along the length of the venules during low flow. We tested the hypothesis that aggregation rarely occurs in the initial 15-microm region of the venule because collision frequency is very low. We found that collision frequency was lower than in other regions, but collision efficiency (the ratio of aggregate formation to collisions) was almost nil in this region, most likely because of entrance effects and time required for aggregation. Radial migration of red blood cells and Dextran 500 had no effect on collision frequency. We also tested the hypothesis that aggregation was reduced in the distal venule region because of the low aggregability of remaining nonaggregated cells. Our findings support this hypothesis, since a simple model based on the ratio of aggregatable to nonaggregatable red blood cells predicts the time course of collision efficiency in this region. Collision efficiency averaged 18% overall but varied from 0 to 52% and was highest in the middle region. We conclude that while collision frequency influences red blood cell aggregate formation in postcapillary venules, collision efficiency is more important.     

Peptides derived from type I thrombospondin repeat-containing proteins of the CCN family inhibit proliferation and migration of endothelial cells

Angiogenesis, or neovascularization, is tightly orchestrated by endogenous regulators that promote or inhibit the process. The fine-tuning of these pro- and anti-angiogenic elements (the angiogenic balance) helps establish the homeostasis in tissues, and any aberration leads to pathologic conditions. The type I thrombospondin repeats are a family of protein structural elements involved in the control of angiogenesis, and some proteins containing these repeats have been identified as negative regulators of angiogenesis. Here we identify a set of 11 novel, anti-angiogenic 18–20-amino acid peptides that are derived from proteins that belong to the CCN protein family and contain type I thrombospondin motifs. We have named these peptides spondinstatin-1, cyrostatin, connectostatin, nephroblastostatin, wispostatin-2, wispostatin-3, netrinstatin-5C, netrinstatin-5D, adamtsostatin-like-4, fibulostatin-6.1, and complestatin-C6 to reflect their origin. We have shown that these peptides inhibit proliferation and migration of human umbilical vein endothelial cells in vitro. By conducting a clustering analysis of the amino acid sequences using sequence similarity criteria and of the experimental results using a hierarchical clustering algorithm, we have demonstrated that there is an underlying correlation between the sequence and activity of the identified peptides. This combination of experimental and computational approaches introduces a novel systematic framework for studying peptide activity, identifying novel peptides with anti-angiogenic activity, and designing mimetic peptides with tailored properties.     

Temporal and spatial variations of cell-free layer width in arterioles

Separation of red blood cells and plasma in microcirculatory vessels produces a cell-free layer at the wall. This layer may be an important determinant of blood viscosity and wall shear stress in arterioles, where most of the hydraulic pressure loss in the circulatory system occurs and flow regulatory mechanisms are prominent. With the use of a newly developed method, the width of the cell-free layer was rapidly and repeatedly determined in arterioles (10- to 50-microm inner diameter) in the rat cremaster muscle at normal arterial pressure. The temporal variation of the cell-free layer width was non-Gaussian, but calculated mean and median values differed by <0.2 microm. The correlation length of the temporal variations downstream (an indication of mixing) was approximately 30 microm and was independent of pseudoshear rate (ratio of mean velocity to vessel diameter) and of vessel diameter. The cell-free layer width was significantly different on opposite sides of the vessel and inversely related. Increasing red blood cell aggregability reduced this inverse relation but had no effect on correlation length. In the diameter range studied, the mean width of the cell-free layer increased from 0.8 to 3.1 microm and temporal variations increased from 30% to 70% of the mean width. Increased aggregability did not alter either relationship. In summary, the cell-free layer width in arterioles is diameter dependent and shows substantial non-Gaussian temporal variations. The temporal variations increase as diameter increases and are inversely related on opposite sides of the vessel.     

Module-based multiscale simulation of angiogenesis in skeletal muscle

Background  

Mathematical modeling of angiogenesis has been gaining momentum as a means to shed new light on the biological complexity underlying blood vessel growth. A variety of computational models have been developed, each focusing on different aspects of the angiogenesis process and occurring at different biological scales, ranging from the molecular to the tissue levels. Integration of models at different scales is a challenging and currently unsolved problem.     

A two-compartment model of VEGF distribution in the mouse

Vascular endothelial growth factor (VEGF) is a key regulator of angiogenesis--the growth of new microvessels from existing microvasculature. Angiogenesis is a complex process involving numerous molecular species, and to better understand it, a systems biology approach is necessary. In vivo preclinical experiments in the area of angiogenesis are typically performed in mouse models; this includes drug development targeting VEGF. Thus, to quantitatively interpret such experimental results, a computational model of VEGF distribution in the mouse can be beneficial. In this paper, we present an in silico model of VEGF distribution in mice, determine model parameters from existing experimental data, conduct sensitivity analysis, and test the validity of the model. The multiscale model is comprised of two compartments: blood and tissue. The model accounts for interactions between two major VEGF isoforms (VEGF(120) and VEGF(164)) and their endothelial cell receptors VEGFR-1, VEGFR-2, and co-receptor neuropilin-1. Neuropilin-1 is also expressed on the surface of parenchymal cells. The model includes transcapillary macromolecular permeability, lymphatic transport, and macromolecular plasma clearance. Simulations predict that the concentration of unbound VEGF in the tissue is approximately 50-fold greater than in the blood. These concentrations are highly dependent on the VEGF secretion rate. Parameter estimation was performed to fit the simulation results to available experimental data, and permitted the estimation of VEGF secretion rate in healthy tissue, which is difficult to measure experimentally. The model can provide quantitative interpretation of preclinical animal data and may be used in conjunction with experimental studies in the development of pro- and anti-angiogenic agents. The model approximates the normal tissue as skeletal muscle and includes endothelial cells to represent the vasculature. As the VEGF system becomes better characterized in other tissues and cell types, the model can be expanded to include additional compartments and vascular elements.     

Intracoronary administration of FGF-2: a computational model of myocardial deposition and retention

This study uses a computational model to characterize the myocardial deposition and retention of basic fibroblast growth factor (FGF-2) at the cellular level after intracoronary (IC) administration of exogenous FGF-2. The model is applied to the in situ conditions present within the myocardium of a dog for which the plasma pharmacokinetics resulting from IC injection of FGF-2 were recorded. Our estimates show that the processes involved in FGF-2 signaling are not diffusion limited; rather, the response time is determined by the reaction time of FGF-2 binding to cell surface receptors. Additionally, the processes of receptor secretion and internalization are found to play crucial roles in the FGF-2 dynamics; future experiments are required to quantify these processes. The model predictions obtained in this study suggest that IC administration of FGF-2 via either a single bolus or repetitive injections causes a transient increase (time scale of hours) in myocardial FGF-2 concentration if the endogenous level of free interstitial FGF-2 is low enough to allow permeation of FGF-2 molecules from the microvascular to the interstitial spaces. The model shows that the majority (64%) of the extracellular FGF-2 ligands are located within the interstitium, and similar fractions are found in the basement membrane and extracellular matrix. Among the FGF-2 molecules found within the interstitium, 2% are free and 98% are bound to interstitial heparan sulfate proteoglycans. These results support the theory of extracellular control of the bioavailability of FGF-2 via dynamic storage of FGF-2 within the basement membrane and extracellular matrix.