Histidine-Rich Glycoprotein Modulates the Anti-Angiogenic Effects of Vasculostatin

Brain angiogenesis inhibitor 1 (BAI1) is a transmembrane protein expressed on glial cells within the brain. Its expression is dramatically down-regulated in many glioblastomas, consistent with its functional ability to inhibit angiogenesis and tumor growth in vivo. We have shown that the soluble anti-angiogenic domain of BAI1 (termed Vstat120) requires CD36, a cell surface glycoprotein expressed on microvascular endothelial cells (MVECs), for it to elicit an anti-angiogenic response. We now report that Vstat120 binding to CD36 on MVECs activates a caspase-mediated pro-apoptotic pathway, and this effect is abrogated by histidine-rich glycoprotein (HRGP). HRGP is a circulating glycoprotein previously shown to function as a CD36 decoy to promote angiogenesis in the presence of thrombospondin-1 or −2. Data here show that Vstat120 specifically binds HRGP. Under favorable MVEC growth conditions this interaction allows chemotactic-directed migration as well as endothelial tube formation to persist in in vitro cellular systems, and increased tumor growth in vivo as demonstrated in both subcutaneous and orthotopic brain tumor models, concomitant with an increase in tumor vascularity. Finally, we show that HRGP expression is increased in human brain cancers, with the protein heavily localized to the basement membrane of the tumors. These data help define a novel angiogenic axis that could be exploited for the treatment of human cancers and other diseases where excess angiogenesis occurs.Angiogenesis is the process through which new blood vessels are formed from pre-existing capillaries. In adulthood the vasculature is normally quiescent, a homeostatic state that is maintained by a precise balance of pro-angiogenic inducers such as vascular endothelial growth factor and anti-angiogenic inhibitors such as thrombospondin (TSP)−1 and −2. This angiostatic balance can be physiologically regulated in favor of new blood vessel formation during processes such as menstruation and wound healing allowing normal tissue regeneration; while pathological disruption of this balance is associated with many disease states, including diabetic retinopathy, atherosclerosis-induced tissue ischemia, chronic inflammation, tumor growth and metastasis, obesity, asthma, and several autoimmune diseases.¹ Therefore, it is extremely important to identify and dissect the pathways involved in vessel growth in both normal and aberrant conditions.Neovascularization is a major component of malignant tumor growth and many therapeutic strategies have been developed to inhibit tumor angiogenesis, including antibodies or decoys that bind and neutralize vascular endothelial growth factor,^2,3 small molecule inhibitors of growth factor signaling pathways,⁴ and peptides based on the anti-angiogenic type I repeat domains (TSR) of TSP-1.^5,6,7 Studies on the mechanisms of TSP-mediated anti-angiogenesis revealed that the TSR domains play an essential role⁸ and that the type B scavenger receptor CD36 functions as the critical endothelial cell surface receptor.^9,10 Using mouse corneal pocket angiogenesis assays we recently demonstrated that CD36 also functions as the receptor for a 120 kDa anti-angiogenic fragment derived from an unrelated TSR-containing protein, Brain Angiogenesis Inhibitor 1 (BAI1). This fragment, known as vasculostatin or Vstat120, suppressed neovessel formation in corneas from wild-type mice yet no effect was observed in CD36 null animals, showing for the first time that a TSR-containing protein distinct from TSP-1 and −2 mediates its anti-angiogenic functions through interactions with CD36.¹¹BAI1 is a 1584-aa brain-specific protein predicted to have seven transmembrane segments and a large extracellular domain. The extracellular domain contains an RGD integrin recognition motif, a putative hormone receptor (HomR) domain, and five TSR domains. Its expression is down-regulated in glioblastomas¹² and inversely correlated with vascularity and metastasis in colorectal cancer,¹³ consistent with an anti-angiogenic role. Kaur et al¹⁴ have shown that the TSR-containing fragment, Vstat120, was released from the cell membrane via proteolytic cleavage at a G protein–coupled receptor cleavage site and that this fragment inhibited microvascular endothelial cell (MVEC) proliferation, migration, and tube formation equivalent to that seen with full-length BAI1. Strikingly, restoration of Vstat120 expression in human glioma cells suppressed tumorigenicity and vascularity and enhanced animal survival in subcutaneous and orthotopic tumor implantation models in nude mice.¹¹The binding site on CD36 for the TSR domains of TSP-1 and −2 and Vstat120 has been localized to amino acids 93 to 120^11,15,16 within a highly conserved region termed the CLESH domain (CD36, LIMP2, EMP structural homology – see Figure 1A). CLESH domains are also found in proteins genetically distinct from the CD36 family,¹⁷ including some, such as HIV gp120 and histidine-rich glycoprotein (HRGP) which are known to bind TSP-1. HRGP, a 75-kDa glycoprotein synthesized in the liver, circulates in the blood at moderately high concentrations (approximately 100 to 150 μg/ml), and is secreted from activated platelets. In addition to TSP-1 and −2, it binds a wide range of ligands including divalent metal cations and several components of the provisional matrix laid down by healing wounds and tumors where angiogenesis is prevalent (for review see¹⁸). It has been implicated in the coagulation and fibrinolytic systems, and high levels have been associated with thrombotic disorders.^19,20 Work in our lab has shown that HRGP acts as a CD36 decoy to promote angiogenesis by binding TSP-1 and −2 and thereby preventing them from binding to CD36 on the endothelial cell surface.^21,22 Because both HRGP and CD36 compete for the identical binding region in TSP-1 and −2, we hypothesized that the relative concentrations of ligands, receptor, and decoy within a given microenvironment would ultimately determine the degree of angiogenesis. Within tumors and other areas of chronic inflammation, HRGP can be released from activated platelets as well as deposited from plasma that gained access via poorly organized hyperpermeable vessels. The angiogenic balance would then be tipped to favor angiogenesis by HRGP binding to TSPs as well as potentially other TSR containing anti-angiogenic proteins.Open in a separate windowFigure 1HRGP CLESH domain fusion protein specifically precipitates Vstat120 from tumor cell postculture media. A: HRGP/GST fusion proteins. Three recombinant GST fusion proteins were designed using fragments of the HRGP protein spanning amino acids 155 to 213 (within the second cystatin domain), amino acids 330 to 389 (the HRR), and amino acids 428 to 502 (CLESH domain). The linear structure of CD36 with the CLESH domain highlighted is shown below for reference. B: Coomassie stained gel (top panel) showing the purified GST-HRGP protein fragments after glutathione sepharose chromatography. The bottom panel shows Western blot analysis of each fusion protein probed with anti-GST antibody. C: Western blot analysis of a GST pull-down assay. The three GST-HRGP fusion proteins and GST alone were bound to glutathione sepharose beads, and CM from LN229V120 glioma cells were added to each sample. Each precipitate was then probed using the Vstat120/BAI1-specific antibody. Vstat120 was only detected when the CLESH domain (HRGP 428 to 502) was used in the pull-down and not the cystatin (HRGP 155 to 213) or the HRR (HRGP 330 to 389) domains. HRGP 428 to 502 only precipitated Vstat120 when CM from Vstat120-transfected LN229 cells (+ lanes) was used. No protein was detected when CM from the nontransfected parent cells (− lane) was used. This is a representative image from n = 3 experiments.In this article we show that the CLESH domain of HRGP binds Vstat120 and suppresses its anti-angiogenic activity by reversing inhibition of endothelial cell migration and tube formation. Furthermore, we show in both subcutaneous and orthotopic brain tumor models that HRGP exacerbates glioblastoma tumor growth and enhances tumor vascularity. We also show that the amount of HRGP present in human brain is increased in patients with primary tumors with the protein localized predominantly within the basement membrane. Finally, we offer some insight into the mechanism of action of Vstat120 by showing caspase-3 activation and endothelial cell apoptosis on Vstat120 addition. Together these results suggest that deposition of HRGP into angiogenic microenvironments, perhaps as the result of the inherent “leakiness” of the neo-vasculature and/or of platelet granule release, can modulate the anti-angiogenic processes mediated by the general family of TSR-containing proteins, and may shed light on the mechanism of angiogenesis regulation in the brain.     

Skin injury following contact with a complex amine

Eight patients with skin damage due to contact with an amine are described. The material is used in the potash industry under the commercial name of Armine to prevent bosselation. Although it is considered to be a strong base, no deep burns were encountered. The reaction to this material initially resembled a dermatitis; later the wound developed the typical appearance of a burn. In addition to the common conservative treatment, antihistamine drugs were used. When a true allergic reaction was suspected steroids were added to the regimen.     

Moderate alcohol intake and renal function decline in women: a prospective study.

BACKGROUND: The impact of moderate alcohol consumption on renal function has important public health implications given the high prevalence of alcohol use. Experimentally, alcohol may adversely affect renal function, but clinical data are limited and no large, prospective studies have examined this issue. METHODS: In a prospective study of 1658 nurses enrolled in the Nurses' Health Study, we sought to determine if there was an association between moderate alcohol consumption and rate of decline in renal function. Daily alcohol intake was measured in 1990, 1994 and 1998 using a detailed questionnaire. Maximum daily alcohol intake was measured in 1988. Creatinine, measured from blood samples provided in 1989 and 2000, was used to estimate glomerular filtration rate (GFR) and creatinine clearance (CCr). RESULTS: Compared to individuals with no alcohol intake, the odds ratios (ORs) for developing a >or=25% estimated GFR decline were: 0.98 (95% CI: 0.72-1.32) for 0.1-4.9 g/day, 0.83 (95% CI: 0.56-1.21) for 5-14.9 g/day and 0.81 (95% CI: 0.50-1.31) for 15-59.9 g/day. For women with hypertension (n = 726), the ORs for a >or=25% estimated GFR decline were: 0.98 (95% CI: 0.53-1.21) for 0.1-4.9 g/day, 0.62 (95% CI: 0.34-1.12) for 5-14.9 g/day and 0.53 (95% CI: 0.25-1.12) for 15-59.9 g/day. CONCLUSIONS: Moderate alcohol consumption had no substantial adverse effect on renal function in women over an 11 year follow-up period.     

Generic products of antiepileptic drugs: A perspective on bioequivalence and interchangeability

Most antiepileptic drugs (AEDs) are currently available as generic products, yet neurologists and patients are reluctant to switch to generics. Generic AEDs are regarded as bioequivalent to brand AEDs after meeting the average bioequivalence criteria; consequently, they are considered to be interchangeable with their respective brands without loss of efficacy and safety. According to the U.S. Food and Drug Administration (FDA) the present bioequivalence requirements are already so rigorous and constrained that there is little possibility that generics that meet regulatory bioequivalence criteria could lead to therapeutic problems. So is there a scientific rationale for the concerns about switching patients with epilepsy to bioequivalent generics? Herein we discuss the assessment of bioequivalence and propose a scaled‐average bioequivalence approach where scaling of bioequivalence is carried out based on brand lot‐to‐lot variance as an alternative to the conventional bioequivalence test as a means to determine whether switching patients to generic formulations, or vice versa, is a safe and effective therapeutic option. Meeting the proposed scaled‐average bioequivalence requirements will ensure that when an individual patient is switched, he or she has fluctuations in plasma levels similar to those from lot‐to‐lot of the brand reference levels and thus should make these generic products safely switchable without change in efficacy and safety outcomes.     

Enuresis in hyperthyroidism: a temporary lack of central control mechanism leads to nocturnal enuresis

We report on a 9-year-old boy who suffered from hyperthyroidism and a new appearance of enuresis. Bedwetting ceased and prepulse inhibition (PPI) – measured as a parameter of central control – increased during the course of therapy.
Conclusion:  The increase in PPI is an indication that enuresis in hyperthyroidism could be as a result of a temporary loss of central control on brainstem reflexes. The case conveys new insights into the correlation between thyroid hormones and micturition patterns and the aetiology of enuresis.     

Prostate cancer risk and ESR1 TA, ESR2 CA repeat polymorphisms.

BACKGROUND: Experimental evidence has suggested that estrogen receptor alpha (coded by the gene ESR1) might increase prostate cancer risk, whereas estrogen receptor beta (coded by the gene ESR2) might reduce prostate cancer risk. METHODS: We investigated the relationship with prostate cancer risk of both a TA repeat polymorphism in the ESR1 5' region, ESR1 (TA)(n), and with a CA repeat polymorphism in intron 5 of ESR2, ESR2 (CA)(n), in a case-control study (545 cases and 674 controls) nested in the Physicians' Health Study. RESULTS: Prostate cancer risk was highest for carriers of ESR1 (TA)(24) and ESR1 (TA)(25). Replacing one modal ESR1 (TA)(14) allele with one ESR1 (TA)(24) allele yielded an odds ratio of 1.42 (95% confidence interval, 1.00-2.00; P=0.05). Replacing one ESR1 (TA)(14) allele with one ESR1 (TA)(25) allele yielded an odds ratio of 2.10 (95% confidence interval, 1.15-3.84; P=0.02). ESR2 (CA)(n) showed no effects on prostate cancer risk. CONCLUSIONS: The ESR1 (TA)(n) polymorphism might play a role in prostate cancer risk.     

Prolapsed ureterocele after upper pole heminephrectomy

Prolapse of a ureterocele through the external meatus is uncommon, and a prolapsed ureterocele occurring after upper pole heminephrectomy is extremely rare. We describe such a case, occurring 2 months after surgery. The ureterocele was excised with the upper pole ureteral stump, and the lower pole ureter was reimplanted with a good outcome.