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Pleiotrophin (PTN) is a growth factor with both pro-angiogenic and limited pro-tumorigenic activity. We evaluated the potential for PTN to be used for safe angiogenic gene therapy using the full length gene and a truncated gene variant lacking the domain implicated in tumorigenesis. Mouse myoblasts were transduced to express full length or truncated PTN (PTN or T-PTN), along with a LacZ reporter gene, and injected into mouse limb muscle and myocardium. In cultured myoblasts, PTN was expressed and secreted via the Golgi apparatus, but T-PTN was not properly secreted. Nonetheless, no evidence of uncontrolled growth was observed in cells expressing either form of PTN. PTN gene delivery to myocardium, and non-ischemic skeletal muscle, did not result in a detectable change in vascularity or function. In ischemic hindlimb at 14 days post-implantation, intramuscular injection with PTN-expressing myoblasts led to a significant increase in skin perfusion and muscle arteriole density. We conclude that (1) delivery of the full length PTN gene to muscle can be accomplished without tumorigenesis, (2) the truncated PTN gene may be difficult to use in a gene therapy context due to inefficient secretion, (3) PTN gene delivery leads to functional benefit in the mouse acute ischemic hindlimb model. [ABSTRACT FROM AUTHOR]

The major vascular endothelial growth factor (VEGF) isoforms are splice variants from a single gene that differ in their extent of heparin affinity due to the absence of the heparin binding domain in the smallest isoform (mouse VEGF120, human VEGF121). A long-held assumption that has guided the use of VEGF isoforms clinically has been that their differences in heparin binding dictate their ability to diffuse through tissue, with VEGF121 moving most freely and that the distribution of recombinant VEGF would have therapeutically relevant consequences. To test this assumption, we delivered the genes encoding these isoforms by myoblast-mediated gene transfer, a means of delivering genes to highly localized sites within muscle. Surprisingly, all isoforms induced comparable extremely localized physiological effects. Significantly, irrespective of the isoform delivered, the vessels passing within several micrometers of muscle fibers expressing VEGF displayed sharply delineated changes in morphology. The induction of capillary wrapping around VEGF-producing fibers, and of vascular malformations in the muscle at high levels, did not differ among isoforms. These results indicate that heparin binding is not essential for the localization of VEGF in adult tissue and suggest that the preferential delivery of VEGF121 cDNA for clinical applications may not have a physiological basis. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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%, P < 0.05) 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.Molecular Therapy (2009) 17 7, 1250–1256. doi:10.1038/mt.2009.85 [ABSTRACT FROM AUTHOR]

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 “infarct size.” 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<0.01 for each method) and wall motion score index (r=0.97-0.98, p<0.01 for each). However, the infarct size calculated using AM was significantly smaller than those using the other measurements (average of values obtained for all hearts studied: AM 10.83±6.53% vs. LM 26.72±19.04%, MLM 26.08±20.94%, p<0.01), and the range of values obtained was compressed 0.4-fold, substantially compromising the accuracy of this approach. We conclude that while wall thinning did not invalidate the use of area-based infarct size measurements, it underestimated the extent of the infarct and substantially reduced the sensitivity of this measure compared to measurements based on infarct arc lengths. We further conclude that the estimation of myocardial and infarct midlines provided comparable accuracy to the more cumbersome tracing of epicardial and endocardial lengths and circumferences. These findings demonstrate that the ratio of summed infarct midlines to summed LV midlines is significantly better than infarct area measurements as an assessment of infarct size in cell or gene therapy studies in chronic rodent MI models.Molecular Therapy (2006) 13, S92–S92; doi: 10.1016/j.ymthe.2006.08.265 [ABSTRACT FROM AUTHOR]

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.Methods: MI was surgically induced in adult male mice by performing a mid-LAD ligation. Closed-chest echo-guided injection of cells (5 x 105 in 5 ul or saline control (HBSS) into myocardium at the peri-infarct border zone was performed at day 3 post-MI in the following groups (n=8/group): mouse BMCs (mBMCs) into C57BL/6 mice, human BMCs into immunodeficient SCID mice, mBMCs into SCID, human EPCs into SCID, and HBSS into both C57BL/6 and SCID as controls. Echocardiography was accomplished in a blinded manner with a Vevo 660 system (VisualSonics, Toronto) at baseline, 2 days post-MI (before injection), and at day 28 post-MI.Results: Left ventricular ejection fraction (LVEF) was uniformly reduced from 57.2+/-4.0% to 38.4+/-3.7% (p<0.0001) after MI in all groups. Mice injected with mBMCs showed the best effect with significant improvement in global LVEF (37.8+/-2.3% vs 44.4+/-6.3%, p=0.03), and in regional wall motion index in the infarct area (1.79+/-0.21 vs 1.4+/-0.29, p=0.02) at day 28, and there was a tendency toward less dilation of cardiac dimensions compared with that before injection. The global systolic function of the treatment group injected with mBMCs was significantly better preserved than that of its corresponding control group (44.4+/-6.3% vs 31.1+/-10.4%, p<0.0005). Human EPC injection showed a non-significant trend toward improvement of LVEF and less LV dilation, while the LVEF of its corresponding control group continued to decrease, resulting in a significant difference between the EPC group and its controls at day 28 (41.5 +/-4.3 vs 32.1+/-6.3, p<0.0005).Conclusions: Unfractionated mouse bone marrow cells attenuated ventricular remodeling and partially preserved LV function when delivered several days after surgically-induced MI. However, injection of different cell types affects LV function to varying extents. The percutaneous echo-guided injection approach made it possible to assess therapeutic cell implantation at a clinically relevant time after MI in a mouse model.Molecular Therapy (2006) 13, S175–S175; doi: 10.1016/j.ymthe.2006.08.521 [ABSTRACT FROM AUTHOR]