Milton Merchant

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.—von Degenfeld, G., Banfi, A., Springer, M. L., Wagner, R. A., Jacobi, J., Ozawa, C. R., Merchant, M. J., Cooke, J. P., Blau, H. M. Microenvironmental VEGF distribution is critical for stable and functional vessel growth in ischemia. FASEB J. 20, E2277–E2287 (2006)

Evidence of specialized leukocyte-vascular homing interactions at the maternal/fetal interface.

In normal pregnancy, the maternal immune system fails to reject the fetus or the placenta as an allogeneic graft. We hypothesize that specialized mechanisms of leukocyte recruitment might limit access of circulating maternal immune cells to the maternal / fetal interface. During the critical period of initial trophoblast invasion there is an elegantly orchestrated progression of leukocyte homing events in the decidua basalis, associated with highly regulated expression of vascular addressins and segregation of specialized leukocyte subsets into well‐defined decidual microdomains. Neutrophils are limited to the region of necrosis associated with enzymatic digestion at the leading edge of the invading trophoblast, where an almost linear array of maternal blood vessels displays the neutrophil ligand E‐selectin. Cells with the phenotype of monocytes but expressing α4β7 integrin are localized in the blood vessels of the specialized “vascular zone”, which display the unusual combination of P‐selectin (partially associated with platelets) and the α4β7 ligand mucosal vascular addressin‐1 (MAdCAM‐1). Granulated metrial gland cells (α4+β7–, probably α4β1+) constitute a well‐defined cluster positioned in the central decidua basalis around venules prominently expressing the α4β1 ligand VCAM‐1 (but not MAdCAM‐1). T and B lymphocytes are rare. Our results suggest that selective mechanisms for regulating leukocyte access, associated with microdomain specialization within the decidua basalis, may play a fundamental role in immune regulation during the invasive period of placental development.

Inflammatory prostaglandin E2 signaling in a mouse model of Alzheimer disease.

There is significant evidence for a central role of inflammation in the development of Alzheimer disease (AD). Epidemiological studies indicate that chronic use of nonsteroidal anti‐inflammatory drugs (NSAIDs) reduces the risk of developing AD in healthy aging populations. As NSAIDs inhibit the enzymatic activity of the inflammatory cyclooxygenases COX‐1 and COX‐2, these findings suggest that downstream prostaglandin signaling pathways function in the preclinical development of AD. Here, we investigate the function of prostaglandin E2 (PGE2) signaling through its EP3 receptor in the neuroinflammatory response to Aβ peptide.

Therapeutic angiogenesis due to balanced single-vector delivery of VEGF and PDGF-BB

Therapeutic angiogenesis by delivery of vascular growth factors is an attractive strategy for treating debilitating occlusive vascular diseases, yet clinical trials have thus far failed to show efficacy. As a result, limb amputation remains a common outcome for muscle ischemia due to severe atherosclerotic disease, with an overall incidence of 100 per million people in the United States per year. A challenge has been that the angiogenic master regulator vascular endothelial growth factor (VEGF) induces dysfunctional vessels, if expressed outside of a narrow dosage window. We tested the hypothesis that codelivery of platelet‐derived growth factor‐BB (PDGF‐BB), which recruits pericytes, could induce normal angiogenesis in skeletal muscle irrespective of VEGF levels. Coexpression of VEGF and PDGF‐BB encoded by separate vectors in different cells or in the same cells only partially corrected aberrant angiogenesis. In marked contrast, coexpression of both factors in every cell at a fixed relative level via a single bicistronic vector led to robust, uniformly normal angiogenesis, even when VEGF expression was high and heterogeneous. Notably, in an ischemic hindlimb model, single‐vector expression led to efficient growth of collateral arteries, revascularization, increased blood flow, and reduced tissue damage. Furthermore, these results were confirmed in a clinically applicable gene therapy approach by adenoviral‐mediated delivery of the bicistronic vector. We conclude that coordinated expression of VEGF and PDGF‐BB via a single vector constitutes a novel strategy for harnessing the potency of VEGF to induce safe and efficacious angiogenesis.—Banfi, A., von Degenfeld, G., Gianni‐Barrera, R., Reginato, S., Merchant, M. J., McDonald, D. M., Blau, H. M. Therapeutic angiogenesis due to balanced single‐vector delivery of VEGF and PDGF‐BB. FASEB J. 26, 2486‐2497 (2012). www.fasebj.org

Signaling via the prostaglandin E-2 receptor EP4 exerts neuronal and vascular protection in a mouse model of cerebral ischemia

Stroke is the third leading cause of death in the United States. Fewer than 5% of patients benefit from the only intervention approved to treat stroke. Thus, there is an enormous need to identify new therapeutic targets. The role of inducible cyclooxygenase (COX-2) activity in stroke and other neurologic diseases is complex, as both activation and sustained inhibition can engender cerebral injury. Whether COX-2 induces cerebroprotective or injurious effects is probably dependent on which downstream prostaglandin receptors are activated. Here, we investigated the function of the PGE2 receptor EP4 in a mouse model of cerebral ischemia. Systemic administration of a selective EP4 agonist after ischemia reduced infarct volume and ameliorated long-term behavioral deficits. Expression of EP4 was robust in neurons and markedly induced in endothelial cells after ischemia-reperfusion, suggesting that neuronal and/or endothelial EP4 signaling imparts cerebroprotection. Conditional genetic inactivation of neuronal EP4 worsened stroke outcome, consistent with an endogenous protective role of neuronal EP4 signaling in vivo. However, endothelial deletion of EP4 also worsened stroke injury and decreased cerebral reperfusion. Systemic administration of an EP4 agonist increased levels of activated eNOS in cerebral microvessels, an effect that was abolished with conditional deletion of endothelial EP4. Thus, our data support the concept of targeting protective prostaglandin receptors therapeutically after stroke.

Blockade of SDF-1 after irradiation inhibits tumor recurrences of autochthonous brain tumors in rats.

Background Tumor irradiation blocks local angiogenesis, forcing any recurrent tumor to form new vessels from circulating cells. We have previously demonstrated that the post-irradiation recurrence of human glioblastomas in the brains of nude mice can be delayed or prevented by inhibiting circulating blood vessel–forming cells by blocking the interaction of CXCR4 with its ligand stromal cell-derived factor (SDF)–1 (CXCL12). In the present study we test this strategy by directly neutralizing SDF-1 in a clinically relevant model using autochthonous brain tumors in immune competent hosts. Methods We used NOX-A12, an l-enantiomeric RNA oligonucleotide that binds and inhibits SDF-1 with high affinity. We tested the effect of this inhibitor on the response to irradiation of brain tumors in rat induced by n-ethyl-N-nitrosourea. Results Rats treated in utero with N-ethyl-N-nitrosourea began to die of brain tumors from approximately 120 days of age. We delivered a single dose of whole brain irradiation (20 Gy) on day 115 of age, began treatment with NOX-A12 immediately following irradiation, and continued with either 5 or 20 mg/kg for 4 or 8 weeks, doses and times equivalent to well-tolerated human exposures. We found a marked prolongation of rat life span that was dependent on both drug dose and duration of treatment. In addition we treated tumors only when they were visible by MRI and demonstrated complete regression of the tumors that was not achieved by irradiation alone or with the addition of temozolomide. Conclusions Inhibition of SDF-1 following tumor irradiation is a powerful way of improving tumor response of glioblastoma multiforme.

Inhibition of CXCR7 extends survival following irradiation of brain tumours in mice and rats

Background:In experimental models of glioblastoma multiforme (GBM), irradiation (IR) induces local expression of the chemokine CXCL12/SDF-1, which promotes tumour recurrence. The role of CXCR7, the high-affinity receptor for CXCL12, in the tumour’s response to IR has not been addressed.Methods:We tested CXCR7 inhibitors for their effects on tumour growth and/or animal survival post IR in three rodent GBM models. We used immunohistochemistry to determine where CXCR7 protein is expressed in the tumours and in human GBM samples. We used neurosphere formation assays with human GBM xenografts to determine whether CXCR7 is required for cancer stem cell (CSC) activity in vitro.Results:CXCR7 was detected on tumour cells and/or tumour-associated vasculature in the rodent models and in human GBM. In human GBM, CXCR7 expression increased with glioma grade and was spatially associated with CXCL12 and CXCL11/I-TAC. In the rodent GBM models, pharmacological inhibition of CXCR7 post IR caused tumour regression, blocked tumour recurrence, and/or substantially prolonged survival. CXCR7 expression levels on human GBM xenograft cells correlated with neurosphere-forming activity, and a CXCR7 inhibitor blocked sphere formation by sorted CSCs.Conclusions:These results indicate that CXCR7 inhibitors could block GBM tumour recurrence after IR, perhaps by interfering with CSCs.

Thrombin-cleaved Fragments of Osteopontin Are Overexpressed in Malignant Glial Tumors and Provide a Molecular Niche with Survival Advantage

Background: Osteopontin (OPN) is highly expressed in glioblastoma (GBM) and possesses inflammatory activity modulated by proteolytic cleavage. Results: Cleaved OPN was increased in GBM and led to more adhesion of GBM cells. OPN conferred resistance to apoptosis in GBM cells. Conclusion: Increased osteopontin proteolysis increased GBM cell resistance to apoptosis. Significance: OPN cleavage links coagulation and inflammation providing a favorable niche for GBM development. Osteopontin (OPN), which is highly expressed in malignant glioblastoma (GBM), possesses inflammatory activity modulated by proteolytic cleavage by thrombin and plasma carboxypeptidase B2 (CPB2) at a highly conserved cleavage site. Full-length OPN (OPN-FL) was elevated in cerebrospinal fluid (CSF) samples from all cancer patients compared with noncancer patients. However, thrombin-cleaved OPN (OPN-R) and thrombin/CPB2-double-cleaved OPN (OPN-L) levels were markedly increased in GBM and non-GBM gliomas compared with systemic cancer and noncancer patients. Cleaved OPN constituted ∼23 and ∼31% of the total OPN in the GBM and non-GBM CSF samples, respectively. OPN-R was also elevated in GBM tissues. Thrombin-antithrombin levels were highly correlated with cleaved OPN, but not OPN-FL, suggesting that the cleaved OPN fragments resulted from increased thrombin and CPB2 in this extracellular compartment. Levels of VEGF and CCL4 were increased in CSF of GBM and correlated with the levels of cleaved OPN. GBM cell lines were more adherent to OPN-R and OPN-L than OPN-FL. Adhesion to OPN altered gene expression, in particular genes involved with cellular processes, cell cycle regulation, death, and inflammation. OPN and its cleaved forms promoted motility of U-87 MG cells and conferred resistance to apoptosis. Although functional mutation of the RGD motif in OPN largely abolished these functions, OPNRAA-R regained significant cell binding and signaling function, suggesting that the SVVYGLR motif in OPN-R may substitute for the RGD motif if the latter becomes inaccessible. OPN cleavage contributes to GBM development by allowing more cells to bind in niches where they acquire anti-apoptotic properties.

Metabolic response of glioma to dichloroacetate measured in vivo by hyperpolarized 13C magnetic resonance spectroscopic imaging

BACKGROUND The metabolic phenotype that derives disproportionate energy via glycolysis in solid tumors, including glioma, leads to elevated lactate labeling in metabolic imaging using hyperpolarized [1-(13)C]pyruvate. Although the pyruvate dehydrogenase (PDH)-mediated flux from pyruvate to acetyl coenzyme A can be indirectly measured through the detection of carbon-13 ((13)C)-labeled bicarbonate, it has proven difficult to visualize (13)C-bicarbonate at high enough levels from injected [1-(13)C]pyruvate for quantitative analysis in brain. The aim of this study is to improve the detection of (13)C-labeled metabolites, in particular bicarbonate, in glioma and normal brain in vivo and to measure the metabolic response to dichloroacetate, which upregulates PDH activity. METHODS An optimized protocol for chemical shift imaging and high concentration of hyperpolarized [1-(13)C]pyruvate were used to improve measurements of lactate and bicarbonate in C6 glioma-transplanted rat brains. Hyperpolarized [1-(13)C]pyruvate was injected before and 45 min after dichloroacetate infusion. Metabolite ratios of lactate to bicarbonate were calculated to provide improved metrics for characterizing tumor metabolism. RESULTS Glioma and normal brain were well differentiated by lactate-to-bicarbonate ratio (P = .002, n = 5) as well as bicarbonate (P = .0002) and lactate (P = .001), and a stronger response to dichloroacetate was observed in glioma than in normal brain. CONCLUSION Our results clearly demonstrate for the first time the feasibility of quantitatively detecting (13)C-bicarbonate in tumor-bearing rat brain in vivo, permitting the measurement of dichloroacetate-modulated changes in PDH flux. The simultaneous detection of lactate and bicarbonate provides a tool for a more comprehensive analysis of glioma metabolism and the assessment of metabolic agents as anti-brain cancer drugs.

Metabolite kinetics in C6 rat glioma model using magnetic resonance spectroscopic imaging of hyperpolarized [1‐13C]pyruvate

In addition to an increased lactate‐to‐pyruvate ratio, altered metabolism of a malignant glioma can be further characterized by its kinetics. Spatially resolved dynamic data of pyruvate and lactate from C6‐implanted female Sprague–Dawley rat brain were acquired using a spiral chemical shift imaging sequence after a bolus injection of a hyperpolarized [1‐13C]pyruvate. Apparent rate constants for the conversion of pyruvate to lactate in three different regions (glioma, normal appearing brain, and vasculature) were estimated based on a two‐site exchange model. The apparent conversion rate constant was 0.018 ± 0.004 s−1 (mean ± standard deviation, n = 6) for glioma, 0.009 ± 0.003 s−1 for normal brain, and 0.005 ± 0.001 s−1 for vasculature, whereas the lactate‐to‐pyruvate ratio, the metabolic marker used to date to identify tumor regions, was 0.36 ± 0.07 (mean ± SD), 0.24 ± 0.07, and 0.12 ± 0.02 for glioma, normal brain, and vasculature, respectively. The data suggest that the apparent conversion rate better differentiate glioma from normal brain (P = 0.001, n = 6) than the lactate‐to‐pyruvate ratio (P = 0.02). Magn Reson Med, 2012.