Jassir Witta

Corneal avascularity is due to soluble VEGF receptor-1.

Corneal avascularity—the absence of blood vessels in the cornea—is required for optical clarity and optimal vision, and has led to the cornea being widely used for validating pro- and anti-angiogenic therapeutic strategies for many disorders. But the molecular underpinnings of the avascular phenotype have until now remained obscure and are all the more remarkable given the presence in the cornea of vascular endothelial growth factor (VEGF)-A, a potent stimulator of angiogenesis, and the proximity of the cornea to vascularized tissues. Here we show that the cornea expresses soluble VEGF receptor-1 (sVEGFR-1; also known as sflt-1) and that suppression of this endogenous VEGF-A trap by neutralizing antibodies, RNA interference or Cre-lox-mediated gene disruption abolishes corneal avascularity in mice. The spontaneously vascularized corneas of corn1 and Pax6+/- mice and Pax6+/- patients with aniridia are deficient in sflt-1, and recombinant sflt-1 administration restores corneal avascularity in corn1 and Pax6+/- mice. Manatees, the only known creatures uniformly to have vascularized corneas, do not express sflt-1, whereas the avascular corneas of dugongs, also members of the order Sirenia, elephants, the closest extant terrestrial phylogenetic relatives of manatees, and other marine mammals (dolphins and whales) contain sflt-1, indicating that it has a crucial, evolutionarily conserved role. The recognition that sflt-1 is essential for preserving the avascular ambit of the cornea can rationally guide its use as a platform for angiogenic modulators, supports its use in treating neovascular diseases, and might provide insight into the immunological privilege of the cornea.

DICER1 deficit induces Alu RNA toxicity in age-related macular degeneration

Geographic atrophy (GA), an untreatable advanced form of age-related macular degeneration, results from retinal pigmented epithelium (RPE) cell degeneration. Here we show that the microRNA (miRNA)-processing enzyme DICER1 is reduced in the RPE of humans with GA, and that conditional ablation of Dicer1, but not seven other miRNA-processing enzymes, induces RPE degeneration in mice. DICER1 knockdown induces accumulation of Alu RNA in human RPE cells and Alu-like B1 and B2 RNAs in mouse RPE. Alu RNA is increased in the RPE of humans with GA, and this pathogenic RNA induces human RPE cytotoxicity and RPE degeneration in mice. Antisense oligonucleotides targeting Alu/B1/B2 RNAs prevent DICER1 depletion-induced RPE degeneration despite global miRNA downregulation. DICER1 degrades Alu RNA, and this digested Alu RNA cannot induce RPE degeneration in mice. These findings reveal a miRNA-independent cell survival function for DICER1 involving retrotransposon transcript degradation, show that Alu RNA can directly cause human pathology, and identify new targets for a major cause of blindness.

Chylomicrons promote intestinal absorption of lipopolysaccharides

Recent data suggest that dietary fat promotes intestinal absorption of lipopolysaccharides (LPS) from the gut microflora, which might contribute to various inflammatory disorders. The mechanism of fat-induced LPS absorption is unclear, however. Intestinal-epithelial cells can internalize LPS from the apical surface and transport LPS to the Golgi. The Golgi complex also contains newly formed chylomicrons, the lipoproteins that transport dietary long-chain fat through mesenteric lymph and blood. Because LPS has affinity for chylomicrons, we hypothesized that chylomicron formation promotes LPS absorption. In agreement with our hypothesis, we found that CaCo-2 cells released more cell-associated LPS after incubation with oleic-acid (OA), a long-chain fatty acid that induces chylomicron formation, than with butyric acid (BA), a short-chain fatty acid that does not induce chylomicron formation. Moreover, the effect of OA was blocked by the inhibitor of chylomicron formation, Pluronic L-81. We also observed that intragastric triolein (TO) gavage was followed by increased plasma LPS, whereas gavage with tributyrin (TB), or TO plus Pluronic L-81, was not. Most intestinally absorbed LPS was present on chylomicron remnants (CM-R) in the blood. Chylomicron formation also promoted transport of LPS through mesenteric lymph nodes (MLN) and the production of TNFα mRNA in the MLN. Together, our data suggest that intestinal epithelial cells may release LPS on chylomicrons from cell-associated pools. Chylomicron-associated LPS may contribute to postprandial inflammatory responses or chronic diet-induced inflammation in chylomicron target tissues.

Loss of SPARC-mediated VEGFR-1 suppression after injury reveals a novel antiangiogenic activity of VEGF-A

VEGF-A promotes angiogenesis in many tissues. Here we report that choroidal neovascularization (CNV) incited by injury was increased by excess VEGF-A before injury but was suppressed by VEGF-A after injury. This unorthodox antiangiogenic effect was mediated via VEGFR-1 activation and VEGFR-2 deactivation, the latter via Src homology domain 2-containing (SH2-containing) tyrosine phosphatase-1 (SHP-1). The VEGFR-1-specific ligand placental growth factor-1 (PlGF-1), but not VEGF-E, which selectively binds VEGFR-2, mimicked these responses. Excess VEGF-A increased CNV before injury because VEGFR-1 activation was silenced by secreted protein, acidic and rich in cysteine (SPARC). The transient decline of SPARC after injury revealed a temporal window in which VEGF-A signaling was routed principally through VEGFR-1. These observations indicate that therapeutic design of VEGF-A inhibition should include consideration of the level and activity of SPARC.

Intestinal Epithelial Serum Amyloid A Modulates Bacterial Growth In Vitro and Pro-Inflammatory Responses in Mouse Experimental Colitis

BackgroundSerum Amyloid A (SAA) is a major acute phase protein of unknown function. SAA is mostly expressed in the liver, but also in other tissues including the intestinal epithelium. SAA reportedly has anti-bacterial effects, and because inflammatory bowel diseases (IBD) result from a breakdown in homeostatic interactions between intestinal epithelia and bacteria, we hypothesized that SAA is protective during experimental colitis.MethodsIntestinal SAA expression was measured in mouse and human samples. Dextran sodium sulfate (DSS) colitis was induced in SAA 1/2 double knockout (DKO) mice and in wildtype controls. Anti-bacterial effects of SAA1/2 were tested in intestinal epithelial cell lines transduced with adenoviral vectors encoding the CE/J SAA isoform or control vectors prior to exposure to live Escherichia coli.ResultsSignificant levels of SAA1/SAA2 RNA and SAA protein were detected by in situ hybridization and immunohistochemistry in mouse colonic epithelium. SAA3 expression was weaker, but similarly distributed. SAA1/2 RNA was present in the ileum and colon of conventional mice and in the colon of germfree mice. Expression of SAA3 was strongly regulated by bacterial lipopolysaccharides in cultured epithelial cell lines, whereas SAA1/2 expression was constitutive and not LPS inducible. Overexpression of SAA1/2 in cultured epithelial cell lines reduced the viability of co-cultured E. coli. This might partially explain the observed increase in susceptibility of DKO mice to DSS colitis. SAA1/2 expression was increased in colon samples obtained from Crohns Disease patients compared to controls.ConclusionsIntestinal epithelial SAA displays bactericidal properties in vitro and could play a protective role in experimental mouse colitis. Altered expression of SAA in intestinal biopsies from Crohns Disease patients suggests that SAA is involved in the disease process..

Deficiency of Endogenous Acute-Phase Serum Amyloid A Protects apoE−/− Mice From Angiotensin II–Induced Abdominal Aortic Aneurysm Formation

Objective— Rupture of abdominal aortic aneurysm (AAA), a major cause of death in the aged population, is characterized by vascular inflammation and matrix degradation. Serum amyloid A (SAA), an acute-phase reactant linked to inflammation and matrix metalloproteinase induction, correlates with aortic dimensions before aneurysm formation in humans. We investigated whether SAA deficiency in mice affects AAA formation during angiotensin II (Ang II) infusion. Approach and Results— Plasma SAA increased ≈60-fold in apoE−/− mice 24 hours after intraperitoneal Ang II injection (100 &mgr;g/kg; n=4) and ≈15-fold after chronic 28-day Ang II infusion (1000 ng/kg per minute; n=9). AAA incidence and severity after 28-day Ang II infusion was significantly reduced in apoE−/− mice lacking both acute-phase SAA isoforms (SAAKO; n=20) compared with apoE−/− mice (SAAWT; n=20) as assessed by in vivo ultrasound and ex vivo morphometric analyses, despite a significant increase in systolic blood pressure in SAAKO mice compared with SAAWT mice after Ang II infusion. Atherosclerotic lesion area of the aortic arch was similar in SAAKO and SAAWT mice after 28-day Ang II infusion. Immunostaining detected SAA in AAA tissues of Ang II–infused SAAWT mice that colocalized with macrophages, elastin breaks, and enhanced matrix metalloproteinase activity. Matrix metalloproteinase-2 activity was significantly lower in aortas of SAAKO mice compared with SAAWT mice after 10-day Ang II infusion. Conclusions— Lack of endogenous acute-phase SAA protects against experimental AAA through a mechanism that may involve reduced matrix metalloproteinase-2 activity.

Impact of Obesity on Ulcerative Colitis Phenotype

induced intestinal lesions were examined in rats. The role of intestinal motility, capsaicinsensitive sensory neurons (CSSNs) and nitric oxide (NO) in the effects of the drugs was also examined. METHODS: Male Wistar rats were treated with IND (10 mg/kg, p.o.) after a 16 h-fast (for gastric ulcers) or without fasting (for intestinal ulcers). The animals were autopsied 24 h after IND administration, and the stomach and small intestine were examined for lesions. Intestinal motility was measured using a balloon method in anesthetized rats. RESULTS: 1. Gastric lesions: IND produced many linear lesions in the corpus mucosa, and the lesion index (LI, total length) in the vehicle-treated group was 25.6±5.3 mm (n=7). The LI was significantly (P<0.05) decreased by pretreatment with oral doses (mg/kg) of CIM (100), RAN (30), FAM (10), OPZ (30), LPZ (30) and RPZ (30). 2. Small intestinal lesions: IND produced many lesions in the middle and lower parts of the small intestine. The LI in the vehicle-treated group was 57.0±10.0 mm (n=10). (a) Effects of H2-RAs: CIM, RAN and FAM increased the LI by 94% (P<0.01), 82% (P<0.01), and 68% (P<0.05), respectively. H2-RAs (3-30, i.v.) did not directly affect intestinal motility, but they did increase the motility induced by IND (10, s.c.). (b) Effects of PPIs: OPZ (100) and RPZ (100) significantly (P<0.05) increased the LI by 48% and 33%, respectively, whereas LPZ (30) significantly (P<0.01) decreased the LI by 61%. The inhibitory effect of LPZ was abolished by functional ablation of CSSNs (20, 30 and 50 mg/kg of capsaicin was administered s.c. for 3 consecutive days 2 weeks before the experiment) or pretreatment with L-NAME (a NO synthase inhibitor). CONCLUSIONS: Though H2-RAs and PPIs markedly inhibited the formation of gastric lesions induced by IND, they (except for LPZ) increased the formation of small intestinal lesions. The results suggest that antisecretory drugs aggravate intestinal lesions induced by NSAIDs, probably by increasing the amount of indigestive foods in the intestine and by increasing intestinal motility. LPZ, though having a potent antisecretory action, inhibited the formation of intestinal lesions, and this action can be explained by its protective action on the mucosa via CSSNs and NO.