Vascular endothelial growth factor (VEGF) exerts crucial functions during pathological angiogenesis and normal physiology. We observed increased hematocrit (60-75%) after high-grade inhibition of VEGF by diverse methods, including adenoviral expression of soluble VEGF receptor (VEGFR) ectodomains, recombinant VEGF Trap protein and the VEGFR2-selective antibody DC101. Increased production of red blood cells (erythrocytosis) occurred in both mouse and primate models, and was associated with near-complete neutralization of VEGF corneal micropocket angiogenesis. High-grade inhibition of VEGF induced hepatic synthesis of erythropoietin (Epo, encoded by Epo) >40-fold through a HIF-1alpha-independent mechanism, in parallel with suppression of renal Epo mRNA. Studies using hepatocyte-specific deletion of the Vegfa gene and hepatocyte-endothelial cell cocultures indicated that blockade of VEGF induced hepatic Epo by interfering with homeostatic VEGFR2-dependent paracrine signaling involving interactions between hepatocytes and endothelial cells. These data indicate that VEGF is a previously unsuspected negative regulator of hepatic Epo synthesis and erythropoiesis and suggest that levels of Epo and erythrocytosis could represent noninvasive surrogate markers for stringent blockade of VEGF in vivo.

Systemic, soluble receptor-mediated VEGF inhibition indicates an essential role for endogenous VEGF during postischemic angiogenesis and hindlimb perfusion.

The ability of water to enter the cells of growing hypocotyl tissue was determined in etiolated soybean (Glycine max (L.) Merr.) seedlings. Water uptake was restricted to that for cell enlargement, and the seedlings were kept intact insofar as possible. Tissue water potentials (ψ w) were measured at thermodynamic equilibrium with an isopiestic thermocouple psychrometer. ψ wwas below the water potential of the environment by as much as 3.1 bars when the tissue was enlarging rapidly. However, ψ w was similar to the water potential of the environment when cell enlargement was not occurring. The low ψ w in enlarging tissue indicates that there was a low conductivity for water entering the cells.The ability of water to enter the enlarging cells was defined as the apparent hydraulic conductivity of the tissue (L'p). Despite the low L'p of growing cells, L'p decreased further as cell enlargement decreased when intact hypocotyl tissue was deprived of endogenous auxin (indole-3-acetic acid) by removal of the hypocotyl hook. Cell enlargement resumed and L'p increased when auxin was resupplied exogenously. The auxin-induced increase in L'p was correlated with the magnitude of the growth enhancement caused by auxin, and it was observed during the earliest phase of the growth response to auxin. The increase in L'p appeared to be caused by an increase in the hydraulic conductivity of the cell protoplasm, since other factors contributing to L'p remained constant. The rapidity of the response is consistent with a cellular site of action at the plasmalemma, although other sites are not precluded.Because the experiments involved only short times, auxin-induced changes in cell enlargement could not be attributed to changes in cell osmotic potentials. Neither could they be attributed to changes in turgor, which increased when the rate of enlargement decreased. Rather, auxin appeared to act by altering the extensibility of the cell walls and by simultaneously altering the ability of water to enter the growing cells under a given water potential gradient. The hydraulic conductivity and extensibility of the cell walls appeared to contribute about equally to the control of the growth rate of the hypocotyls.

Outcome of TGFβ1 signaling is context dependent and differs between individuals due to germ-line genetic variation. To explore innate genetic variants that determine differential outcome of reduced TGFβ1 signaling, we dissected the modifier locus Tgfbm3, on mouse chromosome 12. On a NIH/OlaHsd genetic background, the Tgfbm3b C57 haplotype suppresses prenatal lethality of Tgfb1 −/− embryos and enhances nuclear accumulation of mothers against decapentaplegic homolog 2 (Smad2) in embryonic cells. Amino acid polymorphisms within a disintegrin and metalloprotease 17 (Adam17) can account, at least in part, for this Tgfbm3b effect. ADAM17 is known to down-regulate Smad2 signaling by shedding the extracellular domain of TGFβRI, and we show that the C57 variant is hypomorphic for down-regulation of Smad2/3-driven transcription. Genetic variation at Tgfbm3 or pharmacological inhibition of ADAM17, modulates postnatal circulating endothelial progenitor cell (CEPC) numbers via effects on TGFβRI activity. Because CEPC numbers correlate with angiogenic potential, this suggests that variant Adam17 is an innate modifier of adult angiogenesis, acting through TGFβR1. To determine whether human ADAM17 is also polymorphic and interacts with TGFβ signaling in human vascular disease, we investigated hereditary hemorrhagic telangiectasia (HHT), which is caused by mutations in TGFβ/bone morphogenetic protein receptor genes, ENG, encoding endoglin (HHT1), or ACVRL1 encoding ALK1 (HHT2), and considered a disease of excessive abnormal angiogenesis. HHT manifests highly variable incidence and severity of clinical features, ranging from small mucocutaneous telangiectases to life-threatening visceral and cerebral arteriovenous malformations (AVMs). We show that ADAM17 SNPs associate with the presence of pulmonary AVM in HHT1 but not HHT2, indicating genetic variation in ADAM17 can potentiate a TGFβ-regulated vascular disease.

PurposeThe aim of this study was to develop and characterize a novel peptide imaging agent for noninvasive near-infrared fluorescence imaging of protein transport by the lymphatics. An imaging agent consisting of a cyclic albumin-binding domain (cABD) peptide, with sequence, Arg-Leu-Ile-Glu-Asp-Ile-Cys-Leu-Pro-Arg-Trp-Gly-Cys-Leu-Trp-Glu-Asp-Asp-Lys, was conjugated to a near-infrared fluorophore, IRDye800CW, allowing for enhanced vascular uptake, retention, and fluorescence imaging.ProcedureCharacterization of the cABD-IRDye800 peptide conjugate was performed using fluorescence spectroscopy to assess optical properties and SDS-PAGE and Biacore binding assays to determine binding affinity and specificity. Fluorescence imaging of normal C57BL/6 mice was conducted to monitor lymphatic uptake and retention.ResultscABD-IRDye800 exhibited approximately six times greater fluorescent yield and greater stability than indocyanine green, an agent previously used in humans to image lymphatic vasculature. The agent exhibited affinity for albumin with IC50 and Kd in the nanomolar range and demonstrated superior retention characteristics within mouse lymphatics when compared with IRDye800CW.ConclusionscABD-IRDye800 has utility for assessing lymphatic function in mouse models of human lymphatic disease and the potential for use in clinical diagnostic imaging of the lymphatic vasculature.Electronic supplementary materialThe online version of this article (doi:10.1007/s11307-011-0499-x) contains supplementary material, which is available to authorized users.

NIRF molecular imaging may enable real-time and accurate assessment of tumor margins.

No abstract

The sensorimotor performance of the knee joint in 31 subjects who had undergone unilateral anterior cruciate ligament reconstruction at least 5 months previously was tested under three bracing conditions, 1) the DonJoy Legend brace, 2) a mechanical placebo brace, and 3) no brace, in random order. The accuracy of the subjects' ability to reproduce specified knee joint angles was tested as well as the isokinetic performance of their knee muscles at 60 and 180 deg/sec. The results showed that subjects with the brace or placebo brace performed similarly in reproducing the knee joint positions, but both groups performed better than the subjects without a brace. Isokinetic tests revealed no difference among the three groups in extensor and flexor peak torque production at 60 deg/sec or total work done by the extensors and flexors at 60 and 180 deg/sec. These results suggest that knee bracing can improve the static proprioception of the knee joint, but not the muscle contractile function, in subjects with anterior cruciate ligament reconstruction under isokinetic testing conditions. The finding of similar performances for joint angle reproduction in the brace and placebo brace groups suggests that the apparent improvement in proprioception with knee bracing was not due to the mechanical restraining action of the brace.