Activation of Ras in the Vascular Endothelium Induces Brain Vascular Malformations and Hemorrhagic Stroke.

Cerebrovascular malformations (CVMs) affect approximately 3% of the population, risking hemorrhagic stroke, seizures, and neurological deficits. Recently Ras mutations have been identified in a majority of brain arterio-venous malformations. We generated an endothelial-specific, inducible HRASV12 mouse model, which results in dilated, proliferative blood vessels in the brain, blood-brain barrier breakdown, intracerebral hemorrhage, and rapid lethality. Organoid morphogenesis models revealed abnormal cessation of proliferation, abnormalities in expression of tip and stalk genes, and a failure to properly form elongating tubes. These defects were influenced by both hyperactive PI-3' kinase signaling and altered TGF-ß signaling. Several phenotypic changes predicted by the in vitro morphogenesis analysis were validated in the mouse model. These data provide a model of brain vascular malformations induced by mutant Ras and reveal insights into intersecting molecular mechanisms in the pathogenesis of brain vascular malformations.

Fluorescence Phenomena in Nerve-Labeling Styryl-Type Dyes.

Several classes of diversely substituted styryl type dyes have been synthesized with the goal of extending their expected fluorescent properties as much towards red as possible given the constraint that they maintain drug-like properties and retain high affinity binding to their biological target. We report on the synthesis, optical properties of a series of styryl dyes ( d1-d14 ), and the anomalous photophysical behavior of several of these Donor-Acceptor pairs separated by long conjugated π-systems ( d7-d10 ). We further describe an unusual dual emission behavior with two distinct ground state conformers which could be individually excited to locally excited (LE) and twisted intramolecular charge transfer (TICT) excited state in push-pull dye systems ( d7 , d9 and d10 ). Additionally, unexpected emission behavior in dye systems d7 and d8 wherein the amino- derivative d7 displayed a dual emission in polar medium while the N,N-dimethyl derivative d8 and other methylated derivatives d12-d14 showed only LE emission but did not show any TICT emission. Based on photophysical and nerve binding studies, we down selected compounds that exhibited the most robust fluorescent staining of nerve tissue sections. These dyes ( d7 , d9 , and d10 ) were subsequently selected for in-vivo fluorescence imaging studies in rodents using the small animal multispectral imaging instrument and the dual-mode laparoscopic instrument developed in-house.

Identification of the protein target of myelin-binding ligands by immunohistochemistry and biochemical analyses.

The ability to visualize myelin is important in the diagnosis of demyelinating disorders and the detection of myelin-containing nerves during surgery. The development of myelin-selective imaging agents requires that a defined target for these agents be identified and that a robust assay against the target be developed to allow for assessment of structure-activity relationships. We describe an immunohistochemical analysis and a fluorescence polarization binding assay using purified myelin basic protein (MBP) that provides quantitative evidence that MBP is the molecular binding partner of previously described myelin-selective fluorescent dyes such as BMB, GE3082, and GE3111.

Loss of NF1 expression in human endothelial cells promotes autonomous proliferation and altered vascular morphogenesis.

Neurofibromatosis is a well known familial tumor syndrome, however these patients also suffer from a number of vascular anomalies. The loss of NFl from the endothelium is embryonically lethal in mouse developmental models, however little is known regarding the molecular regulation by NF1 in endothelium. We investigated the consequences of losing NF1 expression on the function of endothelial cells using shRNA. The loss of NF1 was sufficient to elevate levels of active Ras under non-stimulated conditions. These elevations in Ras activity were associated with activation of downstream signaling including activation of ERK, AKT and mTOR. Cells knocked down in NF1 expression exhibited no cellular senescence. Rather, they demonstrated augmented proliferation and autonomous entry into the cell cycle. These proliferative changes were accompanied by enhanced expression of cyclin D, phosphorylation of p27(KIP), and decreases in total p27(KIP) levels, even under growth factor free conditions. In addition, NF1-deficient cells failed to undergo normal branching morphogenesis in a co-culture assay, instead forming planar islands with few tubules and branches. We find the changes induced by the loss of NF1 could be mitigated by co-expression of the GAP-related domain of NF1 implicating Ras regulation in these effects. Using doxycycline-inducible shRNA, targeting NF1, we find that the morphogenic changes are reversible. Similarly, in fully differentiated and stable vascular-like structures, the silencing of NF1 results in the appearance of abnormal vascular structures. Finally, the proliferative changes and the abnormal vascular morphogenesis are normalized by low-dose rapamycin treatment. These data provide a detailed analysis of the molecular and functional consequences of NF1 loss in human endothelial cells. These insights may provide new approaches to therapeutically addressing vascular abnormalities in these patients while underscoring a critical role for normal Ras regulation in maintaining the health and function of the vasculature.

Dual-mode laparoscopic fluorescence image-guided surgery using a single camera.

Iatrogenic nerve damage is a leading cause of morbidity associated with many common surgical procedures. Complications arising from these injuries may result in loss of function and/or sensation, muscle atrophy, and chronic neuropathy. Fluorescence image-guided surgery offers a potential solution for avoiding intraoperative nerve damage by highlighting nerves that are otherwise difficult to visualize. In this work we present the development of a single camera, dual-mode laparoscope that provides near simultaneous display of white-light and fluorescence images of nerves. The capability of the instrumentation is demonstrated through imaging several types of in situ rat nerves via a nerve specific contrast agent. Full color white light and high brightness fluorescence images and video of nerves as small as 100 µm in diameter are presented.

Intraoperative fluorescence imaging of peripheral and central nerves through a myelin-selective contrast agent.

PURPOSE: Patients suffer from complications as a result of unintentional nerve damage during surgery. We focus on improving intraoperative visualization of nerves through the use of a targeted fluorophore and optical imaging instrumentation. PROCEDURE: A myelin-targeting fluorophore, GE3111, was synthesized, characterized for its optical and myelin-binding properties using purified myelin basic protein, and evaluated in mice. Additionally, a compact instrument was adapted to visualize nerves. RESULTS: GE3111 was synthesized using a versatile methodology. Its optical properties were sensitive to the local environment both in vitro and in vivo. Following intravenous injection, central and peripheral nerves were visualized, with the kinetics of nerve uptake modifiable depending on the formulation. Fluorescence polarization showed specific and strong binding to purified myelin basic protein. Nerves were visualized in vivo using a dedicated compact imaging device requiring less than 2.5 mW/cm(2) of illumination at 405 nm. CONCLUSIONS: Fluorescence imaging of nerves through myelin showed a potential for use in image-guided surgery. Intraoperative nerve imaging is an example where contrast agent and instrument development come together as a result of clinical need.

Differential interactions of fluorescent agonists and antagonists with the yeast G protein coupled receptor Ste2p.

We describe a rapid method to probe for mutations in cell surface ligand-binding proteins that affect the environment of bound ligand. The method uses fluorescence-activated cell sorting to screen randomly mutated receptors for substitutions that alter the fluorescence emission spectrum of environmentally sensitive fluorescent ligands. When applied to the yeast α-factor receptor Ste2p, a G protein-coupled receptor, the procedure identified 22 substitutions that red shift the emission of a fluorescent agonist, including substitutions at residues previously implicated in ligand binding and at additional sites. A separate set of substitutions, identified in a screen for mutations that alter the emission of a fluorescent α-factor antagonist, occurs at sites that are unlikely to contact the ligand directly. Instead, these mutations alter receptor conformation to increase ligand-binding affinity and provide signaling in response to antagonists of normal receptors. These results suggest that receptor--agonist interactions involve at least two sites, of which only one is specific for the activated conformation of the receptor.

Activation of endothelial ras signaling bypasses senescence and causes abnormal vascular morphogenesis.

Angiogenesis is crucial for embryogenesis, reproduction, and wound healing and is a critical determinant of tumor growth and metastasis. The multifunctional signal transducer Ras is a proto-oncogene and frequently becomes mutated in a variety of human cancers, including angiosarcomas. Regulation of Ras is important for endothelial cell function and angiogenesis. Hyperactivation of Ras is linked with oncogene-induced senescence in many cell types. Given links between vascular malformations and angiosarcoma with activated Ras signaling, we sought to determine the consequence of sustained Ras activation on endothelial cell function. We find that sustained Ras activation in primary endothelial cells leads to prolonged activation of progrowth signaling, accompanied by a senescence bypass, enhanced proliferation, autonomous growth, and increased survival. Moreover, Ras severely compromises the ability of these cells to organize into vascular structures, instead promoting formation of planar endothelial sheets. This abnormal phenotype is regulated by phosphoinositide 3-kinase signaling, highlighting the therapeutic potential of agents targeting this axis in dealing with vascular morphogenic disorders and vascular normalization of tumors.

Role of extracellular charged amino acids in the yeast alpha-factor receptor.

The yeast pheromone receptor, Ste2p, is a G protein coupled receptor that initiates cellular responses to alpha-mating pheromone, a 13 residue peptide that carries a net positive charge at physiological pH. We have examined the role of extracellular charged groups on the receptor in response to the pheromone. Substitutions of Asn or Ala for one extracellular residue, Asp275, affected both pheromone binding and signaling, suggesting that this position interacts directly with ligand. The other seven extracellular acidic residues could be individually replaced by polar residues with no detectable effects on receptor function. However, substitution of Ala for each of these seven residues resulted in impairment of signaling without affecting pheromone binding, implying that the polar nature of these residues promotes receptor activation. In contrast, substitution of Ala for each of the six positively charged residues at the extracellular surface of Ste2p did not affect signaling.

Oligomerization of the yeast alpha-factor receptor: implications for dominant negative effects of mutant receptors.

Oligomerization of G protein-coupled receptors is commonly observed, but the functional significance of oligomerization for this diverse family of receptors remains poorly understood. We used bioluminescence resonance energy transfer (BRET) to examine oligomerization of Ste2p, a G protein-coupled receptor that serves as the receptor for the alpha-mating pheromone in the yeast Saccharomyces cerevisiae, under conditions where the functional effects of oligomerization could be examined. Consistent with previous results from fluorescence resonance energy transfer (Overton, M. C., and Blumer, K. J. (2000) Curr. Biol. 10, 341-344), we detected efficient energy transfer between Renilla luciferase and a modified green fluorescent protein individually fused to truncated alpha-factor receptors lacking the cytoplasmic C-terminal tail. In addition, the low background of the BRET system allowed detection of significant, but less efficient, energy transfer between full-length receptors. The reduced efficiency of energy transfer between full-length receptors does not appear to result from different levels of receptor expression. Instead, attachment of fluorescent reporter proteins to the full-length receptors appears to significantly increase the distance between reporters. Mutations that were previously reported to block dimerization of truncated alpha-factor receptors reduce but do not completely eliminate BRET transfer between receptors. Dominant negative effects of mutant alleles of alpha-factor receptors appear to be mediated by receptor oligomerization since these effects are abrogated by introduction of additional mutations that reduce oligomerization. We find that heterodimers of normal and dominant negative receptors are defective in their ability to signal. Thus, signal transduction by oligomeric receptors appears to be a cooperative process requiring an interaction between functional monomers.

A fluorescent alpha-factor analogue exhibits multiple steps on binding to its G protein coupled receptor in yeast.

The yeast alpha-factor receptor encoded by the STE2 gene is a member of the extended family of G protein coupled receptors (GPCRs) involved in a wide variety of signal transduction pathways. We report here the use of a fluorescent alpha-factor analogue [K(7)(NBD), Nle(12)] alpha-factor (Lys(7) (7-nitrobenz-2-oxa-1,3-diazol-4-yl), norleucine(12) alpha-factor) in conjunction with flow cytometry and fluorescence microscopy to study binding of ligand to the receptor. Internalization of the fluorescent ligand following receptor binding can be monitored by fluorescence microscopy. The use of flow cytometry to detect binding of the fluorescent ligand to intact yeast cells provides a sensitive and reproducible assay that can be conducted at low cell densities and is relatively insensitive to fluorescence of unbound and nonspecifically bound ligand. Using this assay, we determined that some receptor alleles expressed in cells lacking the G protein alpha subunit exhibit a higher equilibrium binding affinity for ligand than the same alleles expressed in isogenic cells containing the normal complement of G protein subunits. On the basis of time-dependent changes in the intensity and shape of the emission spectrum of [K(7)(NBD),Nle(12)] alpha-factor during binding, we infer that the ligand associates with receptors via a two-step process involving an initial interaction that places the fluorophore in a hydrophobic environment, followed by a conversion to a state in which the fluorophore moves to a more polar environment.