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The immunological potential of animal-derived tissues and organs is the critical hurdle to increasing their clinical implementation. Glutaraldehyde-fixation cross-links proteins in xenogeneic tissues (e.g., bovine pericardium) to delay immune rejection, but also compromises the regenerative potential of the resultant biomaterial. Unfixed xenogeneic biomaterials in which xenoantigenicity has been ameliorated and native extracellular matrix (ECM) architecture has been maintained have the potential to overcome limitations of current clinically utilized glutaraldehyde-fixed biomaterials. The objective of this work was to determine how residual antigenicity and ECM architecture preservation modulate recipient immune and regenerative responses towards unfixed bovine pericardium (BP) ECM scaffolds. Disruption of ECM architecture during scaffold generation, with either SDS-decellularization or glutaraldehyde-fixation, stimulated recipient foreign body response and resultant fibrotic encapsulation following leporine subpannicular implantation. Conversely, BP scaffolds subjected to stepwise removal of hydrophilic and lipophilic antigens using amidosulfobetaine-14 (ASB-14) maintained native ECM architecture and thereby avoided fibrotic encapsulation. Removal of hydrophilic and lipophilic antigens significantly decreased local and systemic graft-specific, adaptive immune responses and subsequent calcification of BP scaffolds compared to scaffolds undergoing hydrophile removal only. Critically, removal of antigenic components and preservation of ECM architecture with ASB-14 promoted full-thickness recipient non-immune cellular repopulation of the BP scaffold. Further, unlike clinically utilized fixed BP, ASB-14-treated scaffolds fostered rapid intimal and medial vessel wall regeneration in a porcine carotid patch angioplasty model. This work highlights the importance of residual antigenicity and ECM architecture preservation in modulating recipient immune and regenerative responses towards xenogeneic biomaterial generation.

Decades of research have been undertaken towards the goal of tissue engineering using xenogeneic scaffolds. The primary advantages associated with use of xenogeneic tissue-derived scaffolds for in vitro development of replacement tissues and organs stem from the inherent extracellular matrix (ECM) composition and architecture. Native ECM possesses appropriate mechanical properties for physiological function of the biomaterial and signals for cell binding, growth, and differentiation. Additionally, xenogeneic tissue is readily available. However, translation of xenogeneic scaffold-derived engineered tissues or organs into clinical therapies requires xenoantigenicity of the material to be adequately addressed prior to implantation. Failure to achieve this goal will result in a graft-specific, host immune rejection response, jeopardizing in vivo survival of the resultant scaffold, tissue, or organ. This review explores (1) the appropriateness of scaffold acellularity as an outcome measure for assessing reduction of the immunological barriers to the use of xenogeneic scaffolds for tissue engineering applications and (2) the need for tissue engineers to strive for antigen removal during xenogeneic scaffold generation.

Numerous studies have focused on generation of unfixed bovine pericardium (BP) extracellular matrix (ECM) for clinical application. However, the extent to which maintenance of native ECM niche is capable of directing behavior of repopulating cells remains relatively unexplored. By exploiting the sidedness of BP scaffolds (i.e., serous or fibrous surface), this study aims to determine the effect of ECM niche preservation on cellular repopulation using different scaffold generation methods. BP underwent either sodium dodecyl sulfate (SDS) decellularization or stepwise, solubilization-based antigen removal using amidosulfobetaine-14 (ASB-14). SDS scaffolds were toxic to repopulating human mesenchymal stem cells (hMSC). Scanning electron microscopy revealed distinct surface ultrastructure of ASB-14 scaffolds based on native BP sidedness. Basement membrane structures on the serous side stimulated hMSC cell monolayer formation, whereas fibrous side facilitated cell penetration into scaffold. Additionally, serous side seeding significantly increased hMSC adhesion and proliferation rate compared to the fibrous side. Furthermore, scaffold ECM niche stimulated sidedness dependent differential hMSC human leukocyte antigen expression, angiogenic and inflammatory cytokine secretion. This work demonstrates that ECM scaffold preparation method and preservation of BP side-based niches critically affects in vitro cell growth patterns and behavior, which has implications for use of such ECM biomaterials in clinical practice.

Effective solubilization of proteins by chaotropes in proteomic applications motivates their use in solubilization-based antigen removal/decellularization strategies. A high urea concentration has previously been reported to significantly reduce lipophilic antigen content of bovine pericardium (BP); however, structure and function of the resultant extracellular matrix (ECM) scaffold were compromised. It has been recently demonstrated that in vivo ECM scaffold fate is determined by two primary outcome measures as follows: (1) sufficient reduction in antigen content to avoid graft-specific adaptive immune responses and (2) maintenance of native ECM structural proteins to avoid graft-specific innate responses. In this work, we assessed residual antigenicity, ECM architecture, ECM content, thermal stability, and tensile properties of BP subjected to a gradient of urea concentrations to determine whether an intermediate concentration exists at which both antigenicity and structure-function primary outcome measures for successful in vivo scaffold outcome can simultaneously be achieved. Alteration in tissue structure-function properties at various urea concentrations with decreased effectiveness for antigen removal makes use of urea-mediated antigen removal unlikely to be suitable for functional scaffold generation.

Bone marrow cell treatment has been proposed as a therapy for myocardial infarction, but the optimal timing and number of injections remain unknown.Myocardial infarction was induced in mice followed by ultrasound-guided injection of mouse bone marrow cells at different time points post myocardial infarction (Days 3, 7, and 14) as monotherapy and at Days 3+7 as "double" therapy and at Days 3+7+14 as "triple" therapy. Controls received saline injections at Day 3 and Days 3+7+14. Left ventricular ejection fraction was evaluated post myocardial infarction prior to any therapy and at Day 28. Hearts were analyzed at Day 28 for infarct size and survival of donor cells.Left ventricular ejection fraction decreased from 55.3±0.9% to 37.6±0.6% (P.001) 2 days post myocardial infarction in all groups. Injection of bone marrow cells at Day 3 post myocardial infarction resulted in smaller infarct size (17.8±3.6% vs. 36.6±7.1%; P=.05) and improved LV function (left ventricular ejection fraction 40.3±2.0% vs. 31.1±8.3%; P.05) compared to control. However, delayed therapy at Day 7 or 14 did not. Multiple injections of bone marrow cells, either double therapy or triple therapy, did not result in reduction in infarct size, but led to improvements in left ventricular ejection fraction at Day 28 compared to control (39.9±3.6% and 38.8±5.5% vs. 34.8±5.3%; all P.05). The number of donor cells surviving at Day 28 did not correlate with improvement in left ventricular ejection fraction.Injection of bone marrow cells at Day 3 reduced infarct size and improved left ventricular function. Multiple injections of bone marrow cells had no additive effect. Delaying cell therapy post myocardial infarction resulted in no functional benefit at all. These results will help inform future clinical trials.

Objectives In patients with coronary artery disease (CAD) medically managed according to currently accepted guidelines, we tested whether a 1-month dietary intervention with flavanol-containing cocoa leads to an improvement of endothelial dysfunction and whether this is associated with an enhanced number and function of circulating angiogenic cells (CACs). Background Dietary flavanols can improve endothelial dysfunction. The CACs, also termed endothelial progenitor cells, are critical for vascular repair and maintenance of endothelial function. Methods In a randomized, controlled, double-masked, cross-over trial, 16 CAD patients (64 ± 3 years of age) received a dietary high-flavanol intervention (HiFI [375 mg]) and a macronutrient- and micronutrient-matched low-flavanol intervention (LoFI [9 mg]) twice daily in random order over 30 days. Results Endothelium-dependent vasomotor function, as measured by flow-mediated vasodilation of the brachial artery, improved by 47% in the HiFI period compared with the LoFI period. After HiFI, the number of CD34+/KDR+-CACs, as measured by flow cytometry, increased 2.2-fold as compared with after LoFI. The CAC functions, as measured by the capacity to survive, differentiate, proliferate, and to migrate were not different between the groups. The HiFI led to a decrease in systolic blood pressure (mean change over LoFI: −4.2 ± 2.7 mm Hg), and increase in plasma nitrite level (mean change over LoFI: 74 ± 32 nM). Applying a mixed-effects linear regression model, the results demonstrated a significant increase in flow-mediated vasodilation and a decrease in systolic blood pressure with increasing levels of CD34+/KDR+-CACs. Conclusions Sustained improvements in endothelial dysfunction by regular dietary intake of flavanols are associated with mobilization of functional CACs. (Effect of Cocoa Flavanols on Vascular Function in Optimally Treated Coronary Artery Disease Patients: Interaction Between Endothelial Progenitor Cells, Reactivity of Micro- and Macrocirculation; NCT00553774).

Brief Secondhand Smoke Exposure Depresses Endothelial Progenitor Cells Activity and Endothelial Function: Sustained Vascular Injury and Blunted Nitric Oxide ProductionChristian Heiss, Nicolas Amabi...

Background Although the incidence of acute death related to coronary artery disease has decreased with the advent of new interventional therapies, myocardial infarction remains one of the leading causes of death in the US. Current animal models developed to replicate this phenomenon have been associated with unacceptably high morbidity and mortality. A new model utilizing the first diagonal branch of the left anterior descending artery (D1-LAD) was developed to provide a clinically relevant lesion, while attempting to minimize the incidence of adverse complications associated with infarct creation. Methods Eight Yucatan miniature pigs underwent percutaneous embolization of the D1-LAD via injection of 90 µm polystyrene micro-spheres. Cardiac structure and function were monitored at baseline, immediately post-operatively, and at 8-weeks post-infarct using transthoracic echocardiography. Post-mortem histopathology and biochemical analyses were performed to evaluate for changes in myocardial structure and extracellular matrix (ECM) composition respectively. Echocardiographic data were evaluated using a repeated measures analysis of variance followed by Tukey’s HSD post hoc test. Biochemical analyses of infarcted to non-infarcted myocardium were compared using analysis of variance. Results All eight pigs successfully underwent echocardiography prior to catheterization. Overall procedural survival rate was 83% (5/6) with one pig excluded due to failure of infarction and another due to deviation from protocol. Ejection fraction significantly decreased from 69.7 ± 7.8% prior to infarction to 50.6 ± 14.7% immediately post-infarction, and progressed to 48.7 ± 8.9% after 8-weeks (p = 0.011). Left ventricular diameter in systole significantly increased from 22.6 ± 3.8 mm pre-operatively to 30.9 ± 5.0 mm at 8 weeks (p = 0.016). Histopathology showed the presence of disorganized fibrosis on hematoxylin and eosin and Picro Sirius red stains. Collagen I and sulfated glycosaminoglycan content were significantly greater in the infarcted region than in normal myocardium (p = 0.007 and p = 0.018, respectively); however, pyridinoline crosslink content per collagen I content in the infarcted region was significantly less than normal myocardium (p = 0.048). Conclusion Systolic dysfunction and changes in ECM composition induced via embolization of the D1-LAD closely mimic those found in individuals with chronic myocardial infarction (MI), and represents a location visible without the need for anesthesia. As a result, this method represents a useful model for studying chronic MI. Electronic supplementary material The online version of this article (doi:10.1186/s12967-015-0547-4) contains supplementary material, which is available to authorized users.

The translation of engineered tissues into clinic requires robust monitoring of tissue development, both in vitro and in vivo . Traditional methods are destructive, time- and cost- inefficient, and do not allow time-lapse measurements from the same sample or animal. This study reports on the ability of time-resolved fluorescence and ultrasound measurements for non-destructive characterization of explanted tissue engineered vascular grafts. Results show that TRFS and FLIm are able to assess alterations in luminal composition namely elastin, collagen and cellular content via changes in fluorescence lifetime values between normal and grafted tissue. These observations are complemented by structural changes observed in UBM pertaining to graft integration and neo-intimal and neo-medial thickening. These results encourage the future application of a catheter-based technique that combines these imaging modalities for nondestructive characterization of vascular grafts in vivo.

The ability of residual antigens on decellularized tissue to elicit the immune response upon implantation motivates development of a more rigorous antigen removal (AR) process for xenogeneic scaffold generation. Antigen removal strategies promoting solubilization of hydrophilic proteins (predominantly cytoplasmic) enhance the reduction of hydrophilic antigenicity in bovine pericardium (BP); however, the diversity of protein antigens within a tissue necessitates development of AR strategies capable of addressing a spectrum of protein antigen solubilities. In the present study, methods for promoting solubilization of lipophilic proteins (predominantly membrane) were investigated for their ability to reduce lipophilic antigenicity of BP when applied as a second AR step following our previously described hydrophilic AR method. Bovine pericardium following AR (BP-AR) was assessed for residual hydrophilic and lipophilic antigenicity, removal of known lipophilic xenoantigens, tensile properties, and extracellular matrix structure and composition. Facilitating hydrophile solubilization (using dithiothreitol and potassium chloride) followed by lipophile solubilization (using amidosulfobetaine-14 (ASB-14)), in a two-step sequential, differential AR strategy, significantly reduces residual hydrophilic and lipophilic antigenicity of BP-AR beyond that achieved with either one-step hydrophilic AR or decellularization using 1% (w/v) sodium dodecyl sulfate. Moreover, use of 1% (w/v) ASB-14 for lipophilic AR eliminates the two most critical known barriers to xenotransplantation (galactose-α(1,3)-galactose and major histocompatibility complex I)) from BP-AR without compromising the structure-function properties of the biomaterial. This study demonstrates the importance of a sequential, differential protein solubilization approach to reduce biomaterial antigenicity in the production of a xenogeneic scaffold for heart valve tissue engineering.

Decellularization techniques have been developed in an attempt to reduce the antigenicity of xenogeneic biomaterials, a critical barrier in their use as tissue engineering scaffolds. However, numerous studies have demonstrated inadequate removal and subsequent persistence of antigens in the biomaterial following decellularization, resulting in an immune response upon implantation. Thus, methods to enhance antigen removal (AR) are critical for the use of xenogeneic biomaterials in tissue engineering and regenerative medicine. In the present study, AR methods incorporating protein solubilization principles were investigated for their ability to reduce antigenicity of bovine pericardium (BP) for heart valve tissue engineering. Bovine pericardium following AR (BP-AR) was assessed for residual antigenicity, tensile properties, and extracellular matrix composition. Increasing protein solubility during AR significantly decreased the residual antigenicity of BP-AR-by an additional 80% compared to hypotonic solution or 60% compared to 0.1% (w/v) SDS decellularization methods. Moreover, solubilizing agents have a dominant effect on reducing the level of residual antigenicity of BP-AR beyond that achieved by AR additives alone. Tested AR methods did not compromise the tensile properties of BP-AR compared to native BP. Furthermore, residual cell nuclei did not correlate to residual antigenicity, demonstrating that residual nuclei counts may not be an appropriate indicator of successful AR. In conclusion, AR strategies promoting protein solubilization significantly reduced residual antigens compared to decellularization methods without compromising biomaterial functional properties. This study demonstrates the importance of solubilizing protein antigens for their removal in the generation of xenogeneic scaffolds.

Objective— Circulating angiogenic cells (CACs), also termed endothelial progenitor cells, play an integral role in vascular repair and are functionally impaired in coronary artery disease (CAD). The role of nitric oxide (NO) in CAC function is poorly understood. We hypothesized that CAC migration toward angiogenic signals is modulated by both NO synthase (NOS) expression and functional response to NO. Methods and Results— Similar to endothelial cells, CAC chemotaxis to vascular endothelial growth factor (VEGF) was blocked by inhibition of NOS, phosphatidylinositol 3-kinase, or guanylyl cyclase or by treatment with an NO scavenger. Addition of an NO donor ( S -nitroso- N -acetylpenicillamine) and the NOS substrate l -arginine increased random cell migration (chemokinesis) and enhanced VEGF-dependent chemotaxis. Healthy CACs expressed endothelial NOS, but endothelial NOS was not detected in CAD patient CACs. Both chemokinesis and chemotaxis to VEGF of patient CACs were decreased compared with healthy CACs but were restored to healthy values by S -nitroso- N -acetylpenicillamine. In parallel, CAD patients exhibited lower flow-mediated vasodilation and plasma NO source nitrite than young, healthy subjects, indicating endothelial dysfunction with reduced NO bioavailability. Conclusion— NOS activity is required for CAC chemotaxis. In CAD patients, impairment of NOS expression and NO bioavailability, rather than response to NO, may contribute to dysfunction of CACs and limit their regenerative capacity.

We compared therapeutic benefits of intramyocardial injection of unfractionated bone marrow cells (BMCs) versus BMC extract as treatments for myocardial infarction (MI), using closed-chest ultrasound-guided injection at a clinically relevant time post-MI. MI was induced in mice and the animals treated at day 3 with either: (i) BMCs from green fluorescent protein (GFP)-expressing mice (n = 14), (ii) BMC extract (n = 14), or (iii) saline control (n = 14). Six animals per group were used for histology at day 6 and the rest followed to day 28 for functional analysis. Ejection fraction was similarly improved in the BMC and extract groups versus control (40.6 +/- 3.4 and 39.1 +/- 2.9% versus 33.2 +/- 5.0%, P0.05) with smaller scar sizes. At day 6 but not day 28, both therapies led to significantly higher capillary area and number of arterioles versus control. At day 6, BMCs increased the number of cycling cardiomyocytes (CMs) versus control whereas extract therapy resulted in significant reduction in the number of apoptotic CMs at the border zone (BZ) versus control. Intracellular components within BMCs can enhance vascularity, reduce infarct size, improve cardiac function, and influence CM apoptosis and cycling early after therapy following MI. Intact cells are not necessary and death of implanted cells may be a major component of the benefit.

Recent studies have shown that increases in proteolytic activity are associated with abdominal aortic aneurysms (AAAs). We have studied samples of the dilated aortic wall, taken during corrective surgery for AAAs, in terms of the number, type, and tissue location of connective tissue proteinases and their inhibitors. Five distinct caseinolytic serine proteinases and six gelatinolytic metalloproteinases were resolved by molecular weight by use of sodium dodecyl sulfate-substrate gel electrophoresis. Isoforms of the Mr 92,000 neutrophil gelatinase were identified by immunoprecipitation of biosynthetically labeled organ culture media. About 50% of the total radiolabeled protein secreted by AAA organ cultures was identified as the Mr 30,000 glycoprotein, tissue inhibitor of metalloproteinase (TIMP), by immunoprecipitation. Both TIMP and gelatinase were localized to the vasa vasorum by immunoperoxidase staining. However, interstitial collagenase could not be detected by any method. These results suggest the involvement of the vasa vasorum in the maintenance and possibly the genesis of AAAs.

Pleiotrophin (PTN) is produced under ischemic conditions and has been shown to induce angiogenesis in vivo. We studied whether or not PTN exerts chemotaxis of pro-angiogenic early endothelial progenitor cells (EPCs), a population of circulating cells that have been reported to participate in and stimulate angiogenesis. Chemotaxis of EPCs, isolated from blood of healthy humans (n = 5), was measured in transwell assays. PTN at 10-500 ng/ml elicited dose-dependent chemotaxis of both EPCs and human umbilical vein endothelial cells (HUVECs), but not of human coronary artery smooth muscle cells (CASMCs) and T98G glioblastoma cells that lack PTN receptors. The degree of chemotaxis was comparable to that induced by the angiogenic factors VEGF and SDF-1alpha. Chemotaxis to PTN was blocked by the NOS inhibitors L-NNA and L-NMMA, the NO scavenger PTIO, the phosphoinositide-3 kinase inhibitor wortmannin, and the guanylyl cyclase inhibitor ODQ, suggesting dependence of EPC chemotaxis on these pathways. PTN induced NOS-dependent production of NO to a similar degree as did VEGF, as indicated by the NO indicator DAF-2. PTN increased proliferation in EPCs and HUVECs to a similar extent as VEGF, but did not induce proliferation of CASMCs. While L-NNA abolished PTN-induced migration in EPCs and HUVECs, it did not inhibit PTN- and VEGF-enhanced proliferation and also caused proliferation by itself. These data suggest that PTN may mediate its pro-angiogenic effects by increasing the local number of not only endothelial cells but also early EPCs at angiogenic sites.

This study sought to analyze the effects of acute secondhand smoke (SHS) exposure on the number and function of endothelial progenitor cells (EPCs) over 24 h.Secondhand smoke increases the risk of vascular disease and is a major public health concern, but the mechanism(s) of action are not fully understood.Healthy nonsmokers (age SEM 30.3 +/- 1.3 years, n = 10) were exposed to 30 min of SHS yielding cotinine levels commonly observed in passive smokers and to smokefree air on 2 separate days. Measurements were taken before exposure (baseline), immediately after (0 h), and at 1 h, 2.5 h, and 24 h after. The EPCs (CD133(+)/KDR(+), CD34(+)/KDR(+)) and endothelial microparticles (EMPs: CD31(+)/CD41(-), CD144(+), CD62e(+)) were determined in blood using flow cytometry. The EPC chemotaxis toward vascular endothelial growth factor was measured. Endothelial function was assessed as flow-mediated dilation (FMD) using ultrasound.Secondhand smoke exposure increased EPCs and plasma vascular endothelial growth factor and completely abolished EPC chemotaxis during 24 h after exposure. Secondhand smoke increased EMPs and decreased FMD. Although FMD returned to baseline at 2.5 h, EMPs and vascular endothelial growth factor levels remained elevated at 24 h, suggesting endothelial activation and injury with functional impairment of the vascular endothelium. Exposure to smokefree air had no effect. Incubation of EPCs from nonexposed subjects with plasma isolated from SHS-exposed subjects in vitro decreased chemotaxis by blockade of vascular endothelial growth factor-stimulated nitric oxide production.Brief exposure to real-world levels of SHS leads to sustained vascular injury characterized by mobilization of dysfunctional EPCs with blocked nitric oxide production. Our results suggest that SHS not only affects the vascular endothelium, but also the function of EPCs.

Efficacy of potential treatments for myocardial infarction (MI) is commonly assessed by histological measurement of infarct size in rodent models. In experiments involving an acute MI setting, measurement of the infarcted area in tissue sections of the left ventricle is a standard approach to determine infarct size. This approach has also been used in the chronic infarct setting to measure infarct area several weeks post-MI. We tested the hypothesis that, because wall thinning is known to occur in the chronic setting, the area measurement approach would be less appropriate. We compared infarct measurements in tissue sections based on 1) infarct area, 2) epicardial and endocardial infarct arc lengths, and 3) midline infarct arc length. Infarct sizes from all three measurement approaches correlated significantly with left ventricular ejection fraction and wall motion abnormality. However, the infarct size values derived from the area measurement approach were significantly smaller than those from the other two measurement approaches, and the range of values obtained was compressed 0.4-fold. The midline method allowed detection of the expected size differences between infarcts of variable severity resulting from proximal vs. distal ligation of the coronary artery. Segmental infarct size was correlated with segmental wall motion abnormality. We conclude that both area- and length-based measurements can be used to determine relative infarct size over a wide range of severity, although the area-based measurements are substantially more compressed due to wall thinning, and that the estimation of infarct midlines is a simple, reliable approach to infarct size assessment.

Extravasation of plasma proteins is increased after volume expansion with whole blood or plasma. To investigate the mechanisms responsible for this phenomenon, we measured extravascular accumulation of exogenous 131I-labeled bovine serum albumin in several tissues and organs of anesthetized rats. Plasma volume was increased acutely by infusion of isoncotic albumin or polyvinylpyrrolidone, with or without subsequent infusion of a 1:10 dilution of the colloid to induce blood-to-tissue fluid movement. Controls were given only a slow sustaining infusion of saline. The amounts of fluid and plasma protein lost from the circulation were followed simultaneously by two methods: 1) material balance in the whole animal, and 2) changes in 131I-labeled albumin uptake (VA) and water content (VW) in the individual tissues. Plasma volume expansion of 80-90% increased plasma protein extravasation in the whole rat by an average of 2.7-fold over a 30-min period. Of the protein extravasated, 42% entered the abdominal cavity. The rest was distributed in the interstitial compartment of various tissues and organs. Tracer albumin accumulation (averaged over 30 min) was increased 38-82% in skin and paw, 40-59% in skeletal muscles, 131% in hearts, and 167-230% in different parts of the intestine. Increased convective transport does not appear to be a major factor. There was little or no relation of albumin transport increase to the magnitude or direction of net fluid transfer. Coupling of albumin transport to volume flow was not greater than previously reported for saline infusion or venous congestion. Convective redistribution (convective transport without net fluid transfer, “volume recirculation”) is estimated to increase albumin transport no more than 10% under the conditions of our experiments. The greater part of the increase is thus dissipative, i.e., attributable to increased diffusion or increased vesicular exchange. Control of dissipative transport of albumin may play an important role in regulating plasma volume.

Assessment of severity of a myocardial infarction (MI) is an important measure of success after experimental delivery of potentially therapeutic genes or cells in rodent MI models. In experiments involving an acute MI setting, histological measurement of the area of the infarcted region in tissue sections of the left ventricle (LV) is a standard assessment of |[ldquo]|infarct size.|[rdquo]| This approach is also used by numerous investigators in a chronic infarct setting, taking infarct area measurements several weeks post-MI. However, we hypothesized that wall thinning due to remodeling of a transmural infarct would invalidate the correlation between a severe infarct and a large infarct scar area measurement, reducing the legitimacy of this approach in a chronic setting. To address this issue, we assessed left ventricular ejection fraction (LVEF; cardiac systolic function) in 8 infarcted mouse hearts and ranked them according to increasing severity. Tissue sections were collected approximately every 300 uM from apex to base and were stained with Masson's trichrome to visualize the infarct scar. The scar was measured in each section by a blinded investigator using several strategies, the numbers obtained were combined to express infarct size as percentage of total LV volume, and the values were compared to the functional measurements for each heart. Our measurement strategies consisted of calculation of the ratio of (1) summed infarct areas to summed LV myocardial areas (Area Measurement: AM), (2) summed epicardial and endocardial infarct arc lengths to summed epicardial and endocardial LV circumferences (Length Measurement: LM), and (3) summed midline infarct arc lengths to summed midline LV circumferences (Midline Length Measurement: MLM). Linear correlation analyses showed that infarct size from all three measurement approaches correlated significantly with LVEF (r=0.87, p

Objective: Myocardial regeneration based on stem cell transplantation has emerged as a potential therapeutic approach toward replacing myocardial scar with functioning contractile tissue after myocardial infarction (MI). Considerable interest has focused on bone marrow cells (BMCs) and endothelial progenitor cells (EPCs) because they appear successful in attenuating remodeling following acute MI. However, preclinical experiments toward this goal have been limited to treatment within hours of an MI, in contrast to human trials, in which cell treatment has been performed several days later after patient stabilization and autologous cell harvesting. We sought to compare the therapeutic effects of different types of putative stem and progenitor cells in a mouse model of MI using a novel closed-chest echo-guided injection approach to deliver cells 3 days after infarction.

Anesthetized rats were infused with lactated Ringer solution (LR) at constant rate for 30 or 60 min; delivered volume loads ranged from 0.03 to 0.08 ml/g body wt. Controls were given only a sustaining infusion of saline at 0.002 ml.g-1.h-1. Only 7-14% of the LR remained in the plasma at the end of the infusion; 76-88% entered the interstitial compartment, and 7-17% was excreted. The amount of plasma protein lost from the circulation with the extravasated fluid was studied simultaneously by two methods: 1) material balance in the whole animal and 2) changes in 131I-labeled albumin uptake (VA) and water content (VW) in individual tissues. The extravasation of 0.03-0.06 ml fluid/g body wt (75-160% initial plasma volume) did not significantly increase plasma protein extravasation in the whole rat. Nearly all of the sampled tissues of LR-infused rats had higher VW than controls. Tissue VA tended to increase with VW, but the regression slopes (delta VA/delta VW), a measure of the tracer albumin concentration of capillary filtrate relative to plasma, were low; skin, 0.006; paw, 0.018; skeletal muscles, 0.007; heart, 0.057; jejunum, 0.095; ileum, 0.045; cecum, 0.026; and colon, 0.027. These ratios are consistent with the very small loss of total plasma protein observed and attest to high solvent-drag reflection coefficients (sigma approximately equal to 1 - delta VA/delta VW): greater than 0.98 in capillaries of skeletal muscles, skin, and paw and 0.91-0.97 in heart and intestine.

Tracer uptake studies were carried out in adult female Nagase (NA) strain analbuminemic rats [derived from Sprague-Dawley (SD) stock] and in adult female SD controls to determine the extent to which capillary permeability to plasma proteins is altered in the absence of endogenous albumin. Accessory measurements (arterial pressure, central venous pressure, plasma and interstitial fluid protein concentrations and oncotic pressures, plasma volume, and interstitial fluid volume) confirm the report of Joles et al. [Am. J. Physiol. 257 (Renal Fluid Electrolyte Physiol. 26): F23-F28, 1989] that shows elevated plasma volumes, normal interstitial fluid volumes, nearly normal plasma oncotic pressures (due to elevated globulin concentrations), and lower interstitial fluid oncotic pressures. In skin, skeletal muscles, and heart muscle, clearances of exogenous heterologous (bovine) albumin were 20–40% higher in NA than in SD controls. In intestine, albumin clearances were 20–30% lower. In NA rats blood-to-tissue clearances of heterologous (bovine) immunoglobulin G in skin and heart were higher and in the intestine they were lower than in SD controls; however, clearances in skeletal muscles were not elevated. The differences between NA and SD are small compared with the large increases in macromolecular permeabilities reported by others for organs and single microvessels perfused with albumin-free fluids.(ABSTRACT TRUNCATED AT 250 WORDS)