The cDNAs of two new human membrane-associated aspartic proteases, memapsin 1 and memapsin 2, have been cloned and sequenced. The deduced amino acid sequences show that each contains the typical pre, pro, and aspartic protease regions, but each also has a C-terminal extension of over 80 residues, which includes a single transmembrane domain and a C-terminal cytosolic domain. Memapsin 2 mRNA is abundant in human brain. The protease domain of memapsin 2 cDNA was expressed in Escherichia coli and was purified. Recombinant memapsin 2 specifically hydrolyzed peptides derived from the ␤-secretase site of both the wild-type and Swedish mutant ␤-amyloid precursor protein (APP) with over 60-fold increase of catalytic efficiency for the latter. Expression of APP and memapsin 2 in HeLa cells showed that memapsin 2 cleaved the ␤-secretase site of APP intracellularly. These and other results suggest that memapsin 2 fits all of the criteria of ␤-secretase, which catalyzes the rate-limiting step of the in vivo production of the ␤-amyloid (A␤) peptide leading to the progression of Alzheimer's disease. Recombinant memapsin 2 also cleaved a peptide derived from the processing site of presenilin 1, albeit with poor kinetic efficiency. Alignment of cleavage site sequences of peptides indicates that the specificity of memapsin 2 resides mainly at the S 1 subsite, which prefers small side chains such as Ala, Ser, and Asp. membrane aspartic proteases ͉ Alzheimer's disease M any proteases are intimately involved in the regulation of cellular and physiological functions. There are five well studied human aspartic proteases. Pepsin and gastricsin participate in digestion in the stomach whereas cathepsins D and E function in intracellular protein degradation in lysosomes and endosomes. Human cathepsin D has been implicated in the metastasis of breast cancer and in Alzheimer's disease. Renin catalyzes the conversion of angiotensinogen to angiotensin 1, a clinically important step in hemostasis. The intimate involvement of human aspartic proteases in physiology and disease illustrates their central role in biology and medicine.The emergence of human gene sequences in the expressed sequence tag (EST) database represents an important new resource for identifying novel human enzymes. We report here the cloning of two new human aspartic proteases, memapsin 1 (M1) and memapsin 2 (M2), initially identified from the human EST database. These proteases are unique among aspartic proteases in that they are membrane-anchored. The presence of M2 in the brain led us to test its capacity to hydrolyze the ␤-amyloid precursor protein (APP). Detailed enzymic and cellular studies suggest that M2 fits all of the criteria of ␤-secretase. Like M2, APP is a type I integral transmembrane protein and is known to be processed in vivo at three sites. The evidence suggests that cleavage at the ␣-secretase site by a membraneassociated metalloprotease is a physiological event. This site is located in APP 12 residues away from the luminal surface of the plasma membrane....

Aims/hypothesis Elevated anti-angiogenic factors such as soluble fms-like tyrosine kinase 1 (sFlt1), a soluble form of vascular endothelial growth factor receptor, and endoglin, a co-receptor for TGFβ1, confer high risk of pre-eclampsia in healthy pregnant women. In this multicentre prospective study, we determined levels of these and related factors in pregnant women with type 1 diabetes, a condition associated with a fourfold increase in pre-eclampsia. Methods Maternal serum sFlt1, endoglin, placental growth factor (PlGF) and pigment epithelial-derived factor were measured in 151 type 1 diabetic and 24 healthy non-diabetic women at each trimester and at term. Diabetologia (2009) Results Approximately 22% of the diabetic women developed pre-eclampsia, primarily after their third trimester visit. In women with pre-eclampsia (diabetic pre-eclampsia, n=26) vs those without hypertensive complications (diabetic normotensive, n=95), significant changes in angiogenic factors were observed, predominantly in the early third trimester and prior to clinical manifestation of pre-eclampsia. Serum sFlt1 levels were increased approximately twofold in type 1 diabetic pre-eclampsia vs type 1 diabetic normotensive women at the third trimester visit (p<0.05) and the normal rise of PlGF during pregnancy was blunted (p<0.05). Among type 1 diabetic women, third trimester sFlt1 and PlGF were inversely related (r 2 =42%, p<0.0001). Endoglin levels were increased significantly in the diabetic group as a whole vs the non-diabetic group (p<0.0001). Conclusions/interpretation Higher sFlt1 levels, a blunted PlGF rise and an elevated sFlt1/PlGF ratio are predictive of pre-eclampsia in pregnant women with type 1 diabetes. Elevated endoglin levels in women with type 1 diabetes may confer a predisposition to pre-eclampsia and may contribute to the high incidence of pre-eclampsia in this patient group.

Serum PEDF levels (mean(SD)) were increased in 96 Type 2 diabetic versus 54 non-diabetic subjects; 5.3(2.8) vs. 3.2(2.0)µg/ml,p<0.001. In diabetes, PEDF correlated with BMI, serum creatinine and LDL-cholesterol, but not with other lipids, HbA1c or CRP. PEDF did not differ by drugs, complications, or gender.Pigment epithelium-derived factor (PEDF) is an anti-angiogenic, anti-inflammatory, and antioxidant factor implicated in diabetic complications [1][2][3][4][5][6]. Previously we reported elevated serum PEDF levels in Type 1 diabetic patients with microvascular complications vs. complication-free patients, and associations with BMI, HbA1c, inflammation, triglycerides, renal and vascular dysfunction [2]. We now compare serum PEDF levels in Type 2 diabetic patients with those in non-diabetic subjects, and relate them to age, gender, vascular risk factors, complications, and medications. Research Design and MethodsThe study, which meets Declaration of Helsinki principles, was approved by Ethics Committees, and each participant gave written informed consent. Microvascular complications were defined as laser-treated retinopathy and/or increased albuminuria. Macrovascular disease was defined as clinically evident cardiovascular, cerebrovascular, or peripheral vascular disease. Blood was collected from 96 Type 2 diabetic and 54 non-diabetic subjects. Sixty-three diabetic and 32 non-diabetic subjects were fasting. HbA1c, lipids, and renal function tests were performed by the Clinical Chemistry Department, and serum CRP was quantified by nephelometry (Dade-Behring, Marburg, Germany) with intra-and inter-assay CVs<8%.

Oxidized and/or glycated low-density lipoprotein (LDL) may mediate capillary injury in diabetic retinopathy. The mechanisms may involve pro-inflammatory and pro-oxidant effects on retinal capillary pericytes. In this study, these effects, and the protective effects of pigment epithelium-derived factor (PEDF), were defined in a primary human pericyte model. Human retinal pericytes were exposed to 100 mg/ml native LDL (N-LDL) or heavily oxidized glycated LDL (HOG-LDL) with or without PEDF at 10-160 nM for 24 h. To assess pro-inflammatory effects, monocyte chemoattractant protein-1 (MCP-1) secretion was measured by ELISA, and nuclear factor-kB (NF-kB) activation was detected by immunocytochemistry. Oxidative stress was determined by measuring intracellular reactive oxygen species (ROS), peroxynitrite (ONOO K ) formation, inducible nitric oxide synthase (iNOS) expression, and nitric oxide (NO) production. The results showed that MCP-1 was significantly increased by HOG-LDL, and the effect was attenuated by PEDF in a dose-dependent manner. PEDF also attenuated the HOG-LDL-induced NF-kB activation, suggesting that the inhibitory effect of PEDF on MCP-1 was at least partially through the blockade of NF-kB activation. Further studies demonstrated that HOG-LDL, but not N-LDL, significantly increased ONOO K formation, NO production, and iNOS expression. These changes were also alleviated by PEDF. Moreover, PEDF significantly ameliorated HOG-LDL-induced ROS generation through up-regulation of superoxide dismutase 1 expression. Taken together, these results demonstrate proinflammatory and pro-oxidant effects of HOG-LDL on retinal pericytes, which were effectively ameliorated by PEDF. Suppressing MCP-1 production and thus inhibiting macrophage recruitment may represent a new mechanism for the salutary effect of PEDF in diabetic retinopathy and warrants more studies in future.