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Biomedical subjects

Matthew L Springer

Publications and source records attributed to Matthew L Springer.

12 recordsLinked to original sources
PubMed2006

Microenvironmental VEGF distribution is critical for stable and functional vessel growth in ischemia.

The critical role of vascular endothelial growth factor (VEGF) expression levels in developmental angiogenesis is well established. Nonetheless, the effects of different local (microenvironmental) VEGF concentrations in ischemia have not been studied in the adult organism, and VEGF delivery to patients has been disappointing. Here, we demonstrate the existence of both lower and upper threshold levels of microenvironmental VEGF concentrations for the induction of therapeutic vessel growth in ischemia. In the ischemic hind limb, implantation of myoblasts transduced to express VEGF164 at different levels per cell increased blood flow only moderately, and vascular leakage and aberrant preangiomatous vessels were always induced. When the same total dose was uniformly distributed by implanting a monoclonal population derived from a single VEGF-expressing myoblast, blood flow was fully restored to nonischemic levels, collateral growth was induced, and ischemic damage was prevented. Hemangiomas were avoided and only normal, pericyte-covered vessels were induced persisting over 15 mo. Surprisingly, clones uniformly expressing either lower or higher VEGF levels failed to provide any functional benefit. A biphasic effect of VEGF dose on vessel number and diameter was found. Blood flow was only improved if vessels were increased both in size and in number. Microenvironmental VEGF concentrations determine efficacy and safety in a therapeutic setting.

Animals↗
PubMed2006

Validation of the wall motion score and myocardial performance indexes as novel techniques to assess cardiac function in mice after myocardial infarction.

The aim of this study was to determine the feasibility and accuracy of wall motion score index (WMSI) and myocardial performance index (MPI) for measuring regional and global left ventricular (LV) function with use of high-resolution echocardiography after myocardial infarction (MI) in mice. In 48 mice, myocardial infarction was induced by ligation in the middle of the left anterior descending coronary artery. Echocardiography was performed under anesthesia at baseline and 1 mo after MI. WMSI was analyzed by a 16-segment model on short-axis views, and wall motion was scored as 1 for normal, 2 for hypokinetic, 3 for akinetic, 4 for dyskinetic, and 5 for aneurysmal. WMSI was calculated as the sum of scores divided by the total number of segments. MPI was calculated on the basis of isovolumetric contraction time (IVCT), isovolumetric relaxation time (IVRT), and ejection time (ET): MPI = (IVCT + IVRT)/ET. We measured LV ejection fraction (LVEF), end-systolic and end-diastolic volumes (ESV and EDV), fractional shortening (FS), and infarct size (IS). LVEF at 4 wk after MI was reduced at 32.8 +/- 9.0%. Linear correlation analyses showed that WMSI (1.6 +/- 0.3) correlated with LVEF (r = -0.84, P < 0.0005), FS (r = -0.43, P = 0.003), and IS (34.3 +/- 15.3%, r = 0.86, P < 0.0005). MPI (0.67 +/- 0.09) correlated with LVEF (r = -0.67, P < 0.0005) and IS (r = 0.72, P < 0.0005). MPI also correlated with mitral inflow velocity (r = -0.68, P < 0.0005) and deceleration time (r = -0.42, P = 0.003). Stepwise regression analysis revealed that WMSI was independently associated with IS. IS, FS, mitral inflow velocity, and deceleration time were independent determinants of MPI. In conclusion, echocardiographic assessments of WMSI and MPI in mice are feasible and correlate strongly with two-dimensional measurement of LV function and IS. These novel parameters provide additional noninvasive assessment of regional and global LV function in mice after MI.

Animals↗
PubMed2006

A balancing act: therapeutic approaches for the modulation of angiogenesis.

Therapeutic stimulation of blood vessel growth (angiogenesis) and vessel enlargement (arteriogenesis) has potential in the treatment of ischemic heart and limb diseases. Numerous experiments have been performed involving the delivery of angiogenic factors, or the genes that encode them, to animal disease models, with generally positive results. Although the results of controlled clinical trials have frequently not lived up to expectations, some have demonstrated limited, but significant, improvements. This review covers the transition of therapeutic angiogenesis approaches from preclinical to clinical studies, describes potential problems in the interpretation of the clinical data, and discusses alternative strategies for the treatment of ischemic diseases using endogenous regulation or delivery of progenitor cells.

Angiogenesis Inducing Agents↗
PubMed2005

VEGF-dependent plasticity of fenestrated capillaries in the normal adult microvasculature.

Unlike during development, blood vessels in the adult are generally thought not to require VEGF for normal function. However, VEGF is a survival factor for many tumor vessels, and there are clues that some normal blood vessels may also depend on VEGF. In this study, we sought to identify which, if any, vascular beds in adult mice depend on VEGF for survival. Mice were treated with a small-molecule VEGF receptor (VEGFR) tyrosine kinase inhibitor or soluble VEGFRs for 1-3 wk. Blood vessels were assessed using immunohistochemistry or scanning or transmission electron microscopy. In a study of 17 normal organs after VEGF inhibition, we found significant capillary regression in pancreatic islets, thyroid, adrenal cortex, pituitary, choroid plexus, small-intestinal villi, and epididymal adipose tissue. The amount of regression was dose dependent and varied from organ to organ, with a maximum of 68% in thyroid, but was less in normal organs than in tumors in RIP-Tag2-transgenic mice or in Lewis lung carcinoma. VEGF-dependent capillaries were fenestrated, expressed high levels of both VEGFR-2 and VEGFR-3, and had normal pericyte coverage. Surviving capillaries in affected organs had fewer fenestrations and less VEGFR expression. All mice appeared healthy, but distinct physiological changes, including more efficient blood glucose handling, accompanied some regimens of VEGF inhibition. Strikingly, most capillaries in the thyroid grew back within 2 wk after cessation of treatment for 1 wk. Our findings of VEGF dependency of normal fenestrated capillaries and rapid regrowth after regression demonstrate the plasticity of the adult microvasculature.

Aging↗
PubMed2005

Closed-chest cell injections into mouse myocardium guided by high-resolution echocardiography.

The mouse is an important model for the development of therapeutic stem cell/bone marrow cell implantation to treat ischemic myocardium. However, its small heart size hampers accurate implantation into the left ventricular (LV) wall. Precise injections have required surgical visualization of the heart, which is subject to complications and is impractical for delayed or repeated injections. Furthermore, the thickness of the myocardium is comparable to the length of a needle bevel, so surgical exposure does not prevent inadvertent injection into the LV cavity. We describe the use of high-resolution echocardiography to guide nonsurgical injections accurately into the mouse myocardial wall. We optimized this system by using a mixture of ultrasound contrast and fluorescent microspheres injected into the myocardium, which enabled us to interpret the ultrasound image of the needle during injection. Quantitative dye injection studies demonstrated that guided closed-chest injections and open-chest injections deliver comparable amounts of injectate to the myocardium. We successfully used this system in a mouse myocardial infarction model to target the injection of labeled cells to a region adjacent to the infarct. Intentional injection of tracer into the LV cavity resulted in a small accumulation in the myocardium, suggesting that non-guided cell injections into mouse hearts may appear to be successful even if the majority of the injectate is lost in the chamber. The use of this system will allow more precise cellular implantation into the mouse myocardium by accurately guiding injections to desired locations, confirming successful implantation of cells, in a clinically relevant time frame.

Animals↗
PubMed2004

Microenvironmental VEGF concentration, not total dose, determines a threshold between normal and aberrant angiogenesis.

Use of long-term constitutive expression of VEGF for therapeutic angiogenesis may be limited by the growth of abnormal blood vessels and hemangiomas. We investigated the relationship between VEGF dosage and the morphology and function of newly formed blood vessels by implanting retrovirally transduced myoblasts that constitutively express VEGF164 into muscles of adult mice. Reducing VEGF dosage by decreasing the total number of VEGF myoblasts implanted did not prevent vascular abnormalities. However, when clonal populations of myoblasts homogeneously expressing different levels of VEGF were implanted, a threshold between normal and aberrant angiogenesis was found. Clonal myoblasts that expressed low to medium levels of VEGF induced growth of stable, pericyte-coated capillaries of uniform size that were not leaky and became VEGF independent, as shown by treatment with the potent VEGF blocker VEGF-TrapR1R2. In contrast, clones that expressed high levels of VEGF induced hemangiomas. Remarkably, when different clonal populations were mixed, even a small proportion of cells with high production of VEGF was sufficient to cause hemangioma growth. These results show for the first time to our knowledge that the key determinant of whether VEGF-induced angiogenesis is normal or aberrant is the microenvironmental amount of growth factor secreted, rather than the overall dose. Long-term continuous delivery of VEGF, when maintained below a threshold microenvironmental level, can lead to normal angiogenesis without other exogenous growth factors.

Animals↗
PubMed2003

Localized arteriole formation directly adjacent to the site of VEGF-induced angiogenesis in muscle.

We have shown previously that implantation of myoblasts constitutively expressing the VEGF-A gene into nonischemic mouse skeletal muscle leads to overgrowth of capillary-like blood vessels and hemangioma formation. These aberrant effects occurred directly at the implantation site. We show here that these regions result from angiogenic capillary growth and involve a change in capillary growth pattern and that smooth muscle-coated vessels similar to arterioles form directly adjacent to the implantation site. Myoblasts genetically engineered to produce VEGF were implanted into mouse leg muscles. Implantation sites were surrounded by a zone of dense capillary-sized vessels, around which was a second zone of muscle containing larger, smooth-muscle-covered vessels but few capillaries, and an outer zone of muscle exhibiting normal capillary density. The lack of capillaries in the middle region suggests that the preexisting capillaries adjacent to the implantation site underwent enlargement and/or fusion and recruited a smooth muscle coat. Capillaries at the implantation site were frequently wrapped around VEGF-producing muscle fibers and were continuous with the circulation and were not observed to include bone-marrow-derived endothelial cells. In contrast with the distant arteriogenesis resulting from VEGF delivery described in previous studies, we report here that highly localized arterioles also form adjacent to the site of delivery.

Animals↗
PubMed2003

Myoblast-mediated gene transfer for therapeutic angiogenesis and arteriogenesis.

Therapeutic angiogenesis aims at generating new blood vessels by delivering growth factors such as VEGF and FGF. Clinical trials are underway in patients with peripheral vascular and coronary heart disease. However, increasing evidence indicates that the new vasculature needs to be stabilized to avoid deleterious effects such as edema and hemangioma formation. Moreover, a major challenge is to induce new vessels that persist following cessation of the angiogenic stimulus. Mature vessels may be generated by modulating timing and dosage of growth factor expression, or by combination of 'growth' factors with 'maturation' factors like PDGF-BB, angiopoietin-1 or TGF-beta. Myoblast-mediated gene transfer has unique characteristics that make it a useful tool for studying promising novel approaches to therapeutic angiogenesis. It affords robust and long-lasting expression, and can be considered as a relatively rapid form of 'adult transgenesis' in muscle. The combined insertion of different gene constructs into single myoblasts and their progeny allows the simultaneous expression of different 'growth' and 'maturation' factors within the same cell in vivo. The additional insertion of a reporter gene makes it possible to analyze the phenotype of the vessels surrounding the transgenic muscle fibers into which the myoblasts have fused. The effects of timing and duration of gene expression can be studied by using tetracycline-inducible constructs, and dosage effects by selecting subpopulations consistently expressing distinct levels of growth factors. Finally, the autologous cell-based approach using transduced myoblasts could be an alternative gene delivery system for therapeutic angiogenesis in patients, avoiding the toxicities seen with some viral vectors.

Arteries↗
PubMed2002

Transient production of alpha-smooth muscle actin by skeletal myoblasts during differentiation in culture and following intramuscular implantation.

alpha-smooth muscle actin (SMA) is typically not present in post-embryonic skeletal muscle myoblasts or skeletal muscle fibers. However, both primary myoblasts isolated from neonatal mouse muscle tissue, and C2C12, an established myoblast cell line, produced SMA in culture within hours of exposure to differentiation medium. The SMA appeared during the cells' initial elongation, persisted through differentiation and fusion into myotubes, remained abundant in early myotubes, and was occasionally observed in a striated pattern. SMA continued to be present during the initial appearance of sarcomeric actin, but disappeared shortly thereafter leaving only sarcomeric actin in contractile myotubes derived from primary myoblasts. Within one day after implantation of primary myoblasts into mouse skeletal muscle, SMA was observed in the myoblasts; but by 9 days post-implantation, no SMA was detectable in myoblasts or muscle fibers. Thus, both neonatal primary myoblasts and an established myoblast cell line appear to similarly reprise an embryonic developmental program during differentiation in culture as well as differentiation within adult mouse muscles.

Actins↗
PubMed2002

RIP2, a checkpoint in myogenic differentiation.

Using a subtractive cDNA library hybridization approach, we found that receptor interacting protein 2 (RIP2), a tumor necrosis factor receptor 1 (TNFR-1)-associated factor, is a novel early-acting gene that decreases markedly in expression during myogenic differentiation. RIP2 consists of three domains: an amino-terminal kinase domain, an intermediate domain, and a carboxy-terminal caspase activation and recruitment domain (CARD). In some cell types, RIP2 has been shown to be a potent inducer of apoptosis and an activator of NF-kappa B. To analyze the function of RIP2 during differentiation, we transduced C2C12 myoblasts with retroviral vectors to constitutively produce RIP2 at high levels. When cultured in growth medium, these cells did not show an enhanced rate of proliferation compared to controls. When switched to differentiation medium, however, they continued to proliferate, whereas control cells withdrew from the cell cycle, showed increased expression of differentiation markers such as myogenin, and began to differentiate into multinucleated myotubes. The complete RIP2 protein appeared to be necessary to inhibit myogenic differentiation, since two different deletion mutants lacking either the amino-terminal kinase domain or the carboxy-terminal CARD had no effect. A mutant deficient in kinase activity, however, had effects similar to wild-type RIP2, indicating that phosphorylation was not essential to the function of RIP2. Furthermore, RIP proteins appeared to be important during myogenic differentiation in vivo, as we detected a marked decrease in expression of the RIP2 homolog RIP in several muscle tissues of the dystrophic mdx mouse, a model for continuous muscle degeneration and regeneration. We conclude that RIP proteins can act independently of TNFR-1 stimulation by ligand to modulate downstream signaling pathways, such as activation of NF-kappa B. These results implicate RIP2 in a previously unrecognized role: a checkpoint for myogenic proliferation and differentiation.

Animals↗