Yihai Cao

Angiostatin: A novel angiogenesis inhibitor that mediates the suppression of metastases by a lewis lung carcinoma

The phenomenon of inhibition of tumor growth by tumor mass has been repeatedly studied, but without elucidation of a satisfactory mechanism. In our animal model, a primary tumor inhibits its remote metastases. After tumor removal, metastases neovascularize and grow. When the primary tumor is present, metastatic growth is suppressed by a circulating angiogenesis inhibitor. Serum and urine from tumor-bearing mice, but not from controls, specifically inhibit endothelial cell proliferation. The activity copurifies with a 38 kDa plasminogen fragment that we have sequenced and named angiostatin. A corresponding fragment of human plasminogen has similar activity. Systemic administration of angiostatin, but not intact plasminogen, potently blocks neovascularization and growth of metastases. We here show that the inhibition of metastases by a primary mouse tumor is mediated, at least in part, by angiostatin.

Spinal cord repair in adult paraplegic rats: partial restoration of hind limb function.

Complete spinal cord gaps in adult rats were bridged with multiple intercostal nerve grafts that redirected specific pathways from white to gray matter. The grafted area was stabilized with fibrin glue containing acidic fibroblast growth factor and by compressive wiring of posterior spinal processes. Hind limb function improved progressively during the first 6 months, as assessed by two scoring systems. The corticospinal tract regenerated through the grafted area to the lumbar enlargement, as did several bulbospinal pathways. These data suggest a possible repair strategy for spinal cord injury.

Proteolytic processing regulates receptor specificity and activity of VEGF‐C

The recently identified vascular endothelial growth factor C (VEGF‐C) belongs to the platelet‐derived growth factor (PDGF)/VEGF family of growth factors and is a ligand for the endothelial‐specific receptor tyrosine kinases VEGFR‐3 and VEGFR‐2. The VEGF homology domain spans only about one‐third of the cysteine‐rich VEGF‐C precursor. Here we have analysed the role of post‐translational processing in VEGF‐C secretion and function, as well as the structure of the mature VEGF‐C. The stepwise proteolytic processing of VEGF‐C generated several VEGF‐C forms with increased activity towards VEGFR‐3, but only the fully processed VEGF‐C could activate VEGFR‐2. Recombinant ‘mature’ VEGF‐C made in yeast bound VEGFR‐3 (KD = 135 pM) and VEGFR‐2 (KD = 410 pM) and activated these receptors. Like VEGF, mature VEGF‐C increased vascular permeability, as well as the migration and proliferation of endothelial cells. Unlike other members of the PDGF/VEGF family, mature VEGF‐C formed mostly non‐covalent homodimers. These data implicate proteolytic processing as a regulator of VEGF‐C activity, and reveal novel structure–function relationships in the PDGF/VEGF family.

Angiogenesis inhibited by drinking tea

Consumption of tea has been shown to inhibit the growth of several tumour types in animals, including cancers of the lung and oesophagus. Drinking tea, especially green tea, is also associated with a lower incidence of human cancer. The mechanisms of cancer inhibition are not known, although several hypotheses have been proposed. We investigated whether drinking green tea could suppress angiogenesis, a process of blood-vessel growth required for tumour growth and metastasis. We find that green tea, and one of its components, epigallocatechin-3-gallate (EGCG), significantly prevents the growth of new blood vessels in animals. This finding indicates that drinking tea may be beneficial for the prevention and treatment of angiogenesis-dependent diseases, including cancer and blindness caused by diabetes.

Angiogenic synergism, vascular stability and improvement of hind-limb ischemia by a combination of PDGF-BB and FGF-2

The establishment of functional and stable vascular networks is essential for angiogenic therapy. Here we report that a combination of two angiogenic factors, platelet-derived growth factor (PDGF)-BB and fibroblast growth factor (FGF)-2, synergistically induces vascular networks, which remain stable for more than a year even after depletion of angiogenic factors. In both rat and rabbit ischemic hind limb models, PDGF-BB and FGF-2 together markedly stimulated collateral arteriogenesis after ligation of the femoral artery, with a significant increase in vascularization and improvement in paw blood flow. A possible mechanism of angiogenic synergism between PDGF-BB and FGF-2 involves upregulation of the expression of PDGF receptor (PDGFR)-α and PDGFR-β by FGF-2 in newly formed blood vessels. Our data show that a specific combination of angiogenic factors establishes functional and stable vascular networks, and provides guidance for the ongoing clinical trials of angiogenic factors for the treatment of ischemic diseases.

Inhibitory PAS domain protein is a negative regulator of hypoxia-inducible gene expression.

Alteration of gene expression is a crucial component of adaptive responses to hypoxia. These responses are mediated by hypoxia-inducible transcription factors (HIFs). Here we describe an inhibitory PAS (Per/Arnt/Sim) domain protein, IPAS, which is a basic helix-loop-helix (bHLH)/PAS protein structurally related to HIFs. IPAS contains no endogenous transactivation function but demonstrates dominant negative regulation of HIF-mediated control of gene expression. Ectopic expression of IPAS in hepatoma cells selectively impairs induction of genes involved in adaptation to a hypoxic environment, notably the vascular endothelial growth factor (VEGF) gene, and results in retarded tumour growth and tumour vascular density in vivo. In mice, IPAS was predominantly expressed in Purkinje cells of the cerebellum and in corneal epithelium of the eye. Expression of IPAS in the cornea correlates with low levels of expression of the VEGF gene under hypoxic conditions. Application of an IPAS antisense oligonucleotide to the mouse cornea induced angiogenesis under normal oxygen conditions, and demonstrated hypoxia-dependent induction of VEGF gene expression in hypoxic corneal cells. These results indicate a previously unknown mechanism for negative regulation of angiogenesis and maintenance of an avascular phenotype.

Angiogenesis modulates adipogenesis and obesity

Substantial evidence shows that neoplastic and nonneoplastic tissue growth is dependent on angiogenesis. Neovascularization and adipogenesis are temporally and spatially coupled processes during prenatal life and they continue to reciprocally interact via paracrine signaling systems throughout adult life. Activated adipocytes produce multiple angiogenic factors including leptin, angiopoietins, HGF, GM-CSF, VEGF, FGF-2, and TGF-beta, which either alone or collectively stimulate neovascularization during fat mass expansion. Thus antiangiogenic agents provide a novel therapeutic option for prevention and treatment of human obesity and its related disorders.

Genomic instability in laminopathy-based premature aging

Premature aging syndromes often result from mutations in nuclear proteins involved in the maintenance of genomic integrity. Lamin A is a major component of the nuclear lamina and nuclear skeleton. Truncation in lamin A causes Hutchinson-Gilford progerial syndrome (HGPS), a severe form of early-onset premature aging. Lack of functional Zmpste24, a metalloproteinase responsible for the maturation of prelamin A, also results in progeroid phenotypes in mice and humans. We found that Zmpste24-deficient mouse embryonic fibroblasts (MEFs) show increased DNA damage and chromosome aberrations and are more sensitive to DNA-damaging agents. Bone marrow cells isolated from Zmpste24−/− mice show increased aneuploidy and the mice are more sensitive to DNA-damaging agents. Recruitment of p53 binding protein 1 (53BP1) and Rad51 to sites of DNA lesion is impaired in Zmpste24−/− MEFs and in HGPS fibroblasts, resulting in delayed checkpoint response and defective DNA repair. Wild-type MEFs ectopically expressing unprocessible prelamin A show similar defects in checkpoint response and DNA repair. Our results indicate that unprocessed prelamin A and truncated lamin A act dominant negatively to perturb DNA damage response and repair, resulting in genomic instability which might contribute to laminopathy-based premature aging.

Monitoring Drug Target Engagement in Cells and Tissues Using the Cellular Thermal Shift Assay

Drug Targeting Drug efficacy depends on the extent of binding to a cellular target (often a protein) with adverse effects caused by excessive target binding or by off-target binding. Engagement of a target protein inside cells is influenced by the effective drug concentration and by factors that regulate the protein conformation, making it difficult to predict efficacy based on in vitro affinity studies. Martinez Molina et al. (p. 84) took advantage of the shift in protein thermal stability caused by drug binding to directly monitor target protein-drug interactions in cells. The method was used to monitor drug target engagement in cancer cells and in mouse livers and kidneys. A method to monitor drug binding to proteins in cells may help in target validation and drug optimization. The efficacy of therapeutics is dependent on a drug binding to its cognate target. Optimization of target engagement by drugs in cells is often challenging, because drug binding cannot be monitored inside cells. We have developed a method for evaluating drug binding to target proteins in cells and tissue samples. This cellular thermal shift assay (CETSA) is based on the biophysical principle of ligand-induced thermal stabilization of target proteins. Using this assay, we validated drug binding for a set of important clinical targets and monitored processes of drug transport and activation, off-target effects and drug resistance in cancer cell lines, as well as drug distribution in tissues. CETSA is likely to become a valuable tool for the validation and optimization of drug target engagement.

Leptin induces vascular permeability and synergistically stimulates angiogenesis with FGF-2 and VEGF

Most endocrine hormones are produced in tissues and organs with permeable microvessels that may provide an excess of hormones to be transported by the blood circulation to the distal target organ. Here, we investigate whether leptin, an endocrine hormone, induces the formation of vascular fenestrations and permeability, and we characterize its angiogenic property in the presence of other angiogenic factors. We provide evidence that leptin-induced new blood vessels are fenestrated. Under physiological conditions, capillary fenestrations are found in the leptin-producing adipose tissue in lean mice. In contrast, no vascular fenestrations were detected in the adipose tissue of leptin-deficient ob/ob mice. Thus, leptin plays a critical role in the maintenance and regulation of vascular fenestrations in the adipose tissue. Leptin induces a rapid vascular permeability response when administrated intradermally. Further, leptin synergistically stimulates angiogenesis with fibroblast growth factor (FGF)-2 and vascular endothelial growth factor (VEGF), the two most potent and commonly expressed angiogenic factors. These findings demonstrate that leptin has another new function—the increase of vascular permeability.