A distinctive histidine residue is essential for in vivo glycation-inactivation of human CD59 transgenically expressed in mice erythrocytes: Implications for human diabetes complications.

Clinical and experimental evidences support a link between the complement system and the pathogenesis of diabetes complications. CD59, an extracellular cell membrane-anchored protein, inhibits formation of the membrane attack complex (MAC), the main effector of complement-mediated tissue damage. This complement regulatory activity of human CD59 (hCD59) is inhibited by hyperglycemia-induced ɛ-amino glycation of Lys41 . Biochemical and structural analyses of glycated proteins with known three-dimensional structure revealed that glycation of ɛ-amino lysyl residues occurs predominantly at "glycation motives" that include lysyl/lysyl pairs or proximity of a histidyl residue, in which the imidazolyl moiety is ≈ 5Å from the ɛ-amino group. hCD59 contains a distinctive Lys41 /His44 putative glycation motif within its active site. In a model of transgenic diabetic mice expressing in erythrocytes either the wild type or a H44Q mutant form of hCD59, we demonstrate in vivo that the His44 is required for Lys41 glycation and consequent functional inactivation of hCD59, as evidenced using a mouse erythrocytes hemolytic assay. Since (1) the His44 residue is not present in CD59 from other animal species and (2) humans are particularly prone to develop complications of diabetes, our results indicate that the Lys41 /His44 glycation motif in human CD59 may confer humans a higher risk of developing vascular disease in response to hyperglycemia.

A specific and sensitive assay for blood levels of glycated CD59: a novel biomarker for diabetes.

Increasing evidence links the complement system with complications of human diabetes. The complement regulatory protein CD59, an inhibitor of formation of membrane attack complex (MAC), is inhibited by hyperglycemia-induced glycation fostering increased deposition of MAC, a major effector of complement-mediated tissue damage. CD59, an ubiquitous GPI-anchored membrane protein, is shed from cell membranes by phospholipases generating a soluble form present in blood and urine. We established an enzyme-linked immunosorbent assay (ELISA) to measure serum/plasma glycated human CD59 (hCD59) (GCD59) and evaluated its potential as a diabetes biomarker. We used a synthetic peptide strategy to generate (a) a mouse monoclonal antibody to capture hCD59, (b) a rabbit monoclonal antibody to detect GCD59, and (c) a GCD59 surrogate for assay standardization. ELISA conditions were optimized for precision, reproducibility, and clinical sensitivity. The clinical utility of the assay was initially evaluated in 24 subjects with or without diabetes and further validated in a study that included 100 subjects with and 90 subjects without a diagnosis of diabetes. GCD59 (a) was significantly higher in individuals with than in individual without diabetes, (b) was independently associated with HbA1c, and (c) identified individuals with diabetes with high specificity and sensitivity. We report the development and standardization of a novel, sensitive, and specific ELISA for measuring GCD59 in blood. The assay distinguished individuals with diabetes from those without, and showed strong correlation between GCD59 and HbA1c. Because GCD59 likely contributes to the pathogenesis of diabetes complications, measurement of blood levels of GCD59 may be useful in the diagnosis and management of diabetes.

Expression of PITX2 homeodomain transcription factor during rat gonadal development in a sexually dimorphic manner.

PITX2, a multifunctional Paired-like homeodomain transcription factor, plays obligatory role during development of organs like heart, brain and pituitary. It regulates differentiation of vascular smooth muscle cells and hematopoietic stem cells. Although we earlier reported the Pitx2/PITX2 expression in gonad, but the expression pattern of its different isoforms in mammalian gonads especially during development is still not known. As PITX2 participates in the development of multiple organs and different homeobox genes have been shown to control gonadal functions, we wanted to investigate the role of PITX2 in gonadal development and its function. The objective of our study was to know the expression profile of different Pitx2/PITX2 isoforms and its localization throughout the development of gonads. Here we show the temporal and spatial expression pattern of Pitx2/PITX2 and its localization throughout the embryonic and postnatal stages of rat gonads. Pitx2/PITX2 expression profile reveals the differential and dimorphic expression pattern of its two isoforms PITX2B2 and-Cß throughout the embryonic development stages and also in the postnatal stages, where it becomes more prominent. This is the first report where PITX2 homeodomain transcription factor shows isoform-specific sexually dimorphic expression. In addition, PITX2 localization was found in the embryonic ovarian primordial germ cell clusters and germ cells inside the testicular cords and also in somatic cells. In adults, ovarian granulosa and theca cells as well as germ cells inside the seminiferous tubules in testis express PITX2. All the evidences suggest that the differential expression of PITX2 might be associated with sex-specific embryonic and postnatal gonadal development and the physiological processes.

Deficiency of the complement regulatory protein CD59 accelerates the development of diabetes-induced atherosclerosis in mice.

AIMS: Clinical and experimental evidence supports a strong link between the complement system, complement regulatory proteins and the pathogenesis of diabetes vascular complications. We previously reported that the complement regulatory protein CD59 is inactivated by glycation in humans with diabetes. Our objective for this study is to assess experimentally how the deficiency of CD59 impacts the development of diabetic atherosclerosis in vivo. METHODS: We crossed mCD59 sufficient and deficient mice into the ApoE-/- background to generate mCd59ab+/+/ApoE-/- and mCd59ab-/-/ApoE-/- mice, and induced diabetes by multiple low dose injections of streptozotocin. Atherosclerosis was detected by hematoxylin and eosin (H&E) and oil red-O staining. Membrane attack complex (MAC) deposition and macrophage infiltration were detected by immunostaining. RESULTS: Diabetic mCD59 deficient (mCD59ab-/-/ApoE-/-) mice developed nearly 100% larger atherosclerotic lesion areas in the aorta (7.5%±0.6 vs 3.6%±0.7; p<0.005) and in the aortic roots (H&E: 26.2%±1.9 vs. 14.3%±1.1; p<0.005), in both cases associated with increased lipid (Oil red-O: 14.9%±1.1 vs. 7.8%±1.1; p<0.05) and MAC deposition (6.8%±0.8 vs. 3.0%±0.7; p<0.005) and macrophage infiltration (31.5%±3.7 vs. 16.4%±3.0; p<0.05) in the aortic roots as compared to their diabetic mCD59 sufficient (mCD59ab+/+/ApoE-/-) counterpart. CONCLUSIONS: The deficiency of CD59 accelerates the development of diabetic atherosclerosis.

Plasma Glycated CD59, a Novel Biomarker for Detection of Pregnancy-Induced Glucose Intolerance.

OBJECTIVE: Plasma glycated CD59 (pGCD59) is an emerging biomarker in diabetes. We assessed whether pGCD59 could predict the following: the results of the glucose challenge test (GCT) for screening of gestational diabetes mellitus (GDM) (primary analysis); and the diagnosis of GDM and prevalence of large for gestational age (LGA) newborns (secondary analyses). RESEARCH DESIGN AND METHODS: Case-control study of 1,000 plasma samples from women receiving standard prenatal care, 500 women having a normal GCT (control subjects) and 500 women with a failed GCT and a subsequent oral glucose tolerance test (case patients). RESULTS: Compared with control subjects, the median (interquartile range) pGCD59 value was 8.5-fold higher in case patients and 10-fold higher in GDM patients, as follows: control subjects 0.33 (0.19); case patients 2.79 (1.4); GDM patients 3.23 (1.43) (P < 0.001); area under the receiver operating characteristic curve 0.92. LGA prevalence was 4.3% in the lowest quartile and 13.5% in the highest quartile of pGCD59. CONCLUSIONS: One pGCD59 measurement during weeks 24-28 identifies pregnancy-induced glucose intolerance with high sensitivity and specificity and can potentially identify the risk for LGA.

Differential expression of procollagen lysine 2-oxoglutarate 5-deoxygenase and matrix metalloproteinase isoforms in hypothyroid rat ovary and disintegration of extracellular matrix.

Hypothyroid-induced reproductive malfunction in both the sexes is a common phenomenon of global concern. In an attempt to characterize the differentially expressed genes that might be responsible for these disorders, we have identified a number of clones in hypothyroid rat ovary by subtractive hybridization. One such clone is procollagen lysyl hydroxylase2 (Plod-2), the key enzyme for the first step of collagen biosynthetic pathway, which was down-regulated in hypothyroid condition. We have also demonstrated the reduced expression of other isoforms of Plods, namely Plod-1 and -3 in hypothyroid rat ovary. The current studies are the first of their kind to report that thyroid hormone regulates the Plod gene in rat ovary. Moreover, we have shown the up-regulation of matrix-degrading enzyme(s), matrix metalloproteinase(s) in the hypothyroid rat ovary, whereas the tissue-inhibitory metalloproteinase is down-regulated. Finally, the results of the present studies indicate that in hypothyroid condition, collagen biosynthesis in ovary seems to be disturbed with concomitant enhancement in collagen degradation, resulting in disintegration of overall ovarian structure.

Role of complement and complement regulatory proteins in the complications of diabetes.

It is well established that the organ damage that complicates human diabetes is caused by prolonged hyperglycemia, but the cellular and molecular mechanisms by which high levels of glucose cause tissue damage in humans are still not fully understood. The prevalent hypothesis explaining the mechanisms that may underlie the pathogenesis of diabetes complications includes overproduction of reactive oxygen species, increased flux through the polyol pathway, overactivity of the hexosamine pathway causing intracellular formation of advanced glycation end products, and activation of protein kinase C isoforms. In addition, experimental and clinical evidence reported in past decades supports a strong link between the complement system, complement regulatory proteins, and the pathogenesis of diabetes complications. In this article, we summarize the body of evidence that supports a role for the complement system and complement regulatory proteins in the pathogenesis of diabetic vascular complications, with specific emphasis on the role of the membrane attack complex (MAC) and of CD59, an extracellular cell membrane-anchored inhibitor of MAC formation that is inactivated by nonenzymatic glycation. We discuss a pathogenic model of human diabetic complications in which a combination of CD59 inactivation by glycation and hyperglycemia-induced complement activation increases MAC deposition, activates pathways of intracellular signaling, and induces the release of proinflammatory, prothrombotic cytokines and growth factors. Combined, complement-dependent and complement-independent mechanisms induced by high glucose promote inflammation, proliferation, and thrombosis as characteristically seen in the target organs of diabetes complications.

Glycation of the complement regulatory protein CD59 is a novel biomarker for glucose handling in humans.

CONTEXT: Human CD59, an inhibitor of the membrane attack complex of complement, is inactivated by glycation. Glycation inactivation of CD59 enhances complement-mediated injury in target organs of diabetes complications. OBJECTIVE: We hypothesized that circulating soluble glycated CD59 (GCD59) represents a novel biomarker of blood glucose handling and aimed to conduct human study protocols to test this hypothesis. DESIGN, SETTING, PARTICIPANTS, AND OUTCOME MEASURES: Using a newly developed ELISA, we measured circulating soluble GCD59 in samples from 3 separate human studies evaluating acute and chronic glucose handling and glucose responses to insulin therapy. Study 1 (normal vs diabetic subjects) evaluated the cross-sectional association between GCD59 and glycated hemoglobin (HbA1c) in 400 subjects with and without type 2 diabetes. Study 2 (oral glucose tolerance test [OGTT] in nondiabetics) evaluated whether fasting GCD59 independently predicted the 2-hour glucose response to an OGTT in 109 subjects without a diagnosis of diabetes. Study 3 (intensified insulin treatment) evaluated the effect of intensification of glycemic control with insulin on GCD59 in 21 poorly controlled individuals with diabetes. RESULTS: In study 1 (normal vs diabetic subjects), GCD59 was independently and positively associated with HbA1c in individuals with and without diabetes (ß = 1.1, P < .0001 and ß = 1.1 P < .001, respectively). In study 2 (OGTT in nondiabetics), a single GCD59 measurement independently predicted the results of the 2-hour OGTT (ß = 19.8, P < .05) after multivariate modeling. In study 3 (intensified insulin treatment), intensification of glucose control with insulin resulted in a concomitant and parallel reduction of average weekly glucose and GCD59 within 2 weeks. CONCLUSIONS: We observed robust relationships between a single measurement of blood levels of GCD59 and both acute (2-hour OGTT) and chronic (HbA1c) measures of glucose handling. Lowering of GCD59 levels closely reflected lowering of average weekly glucose within 2 weeks. The role of GCD59 in the diagnosis, management, and vascular risk stratification in diabetes warrants further investigation.

Procollagen synthesis is increased in hypothyroid rat ovary by a parallel and compensatory pathway.

Collagen biosynthesis is a multistep process that starts with the transcription and translation of the individual collagen gene. It is characterized by the presence of a large number of co- and posttranslational modifications. Hydroxylysine is found only in animal proteins and mostly in collagens. Procollagen lysyl hydroxylation is the first step in collagen biosynthetic pathway and lysyl hydroxylases (Plod isoforms) are responsible for this enzymatic process. Previously we showed the down regulation of Plod isoforms in hypothyroid ovary. As hypothyroidism is a stress for normal animals, we wanted to explore whether any compensatory pathway exists to balance the reduced lysyl hydroxylation of collagen in hypothyroid rat ovary. In this report we have shown that procollagen I and III are increased in hypothyroid condition and subsequently decreased upon T(3) add-back. Heat Shock Protein-47 is a collagen-specific molecular chaperone and its existence in ovary has been documented. The genes encoding HSP-47, prolyl-4-hydroxylase-alpha and -beta (P4H-alpha and -beta) are increased in hypothyroid condition. Down regulation of lysyl hydroxylase in hypothyroid condition results less collagen formation. At the same time over production of procollagens, HSP-47 and P4H is very significant as they may compensate the damage whatsoever caused due to hypothyroidism in ovarian tissue.

Localization and thyroid hormone influenced expression of collagen II in ovarian tissue.

Collagen type II (Col II), one of the main components of the hyaline cartilage, is a member of the fibril-forming collagen family. Due to its amino acid composition, the extent of lysine hydroxylation of Col II is much higher than that of other fibril forming collagens. Since lysyl hydroxylase isoforms are less synthesized in hypothyroid ovarian tissue, Col II level is expected to be reduced here and contribute to the degradation of ovarian ECM in this condition. As there was no previous report, we have demonstrated Col II expression in rat ovary. Col2A1 mRNA shares significant part of the total collagens in ovary as shown by the relative expression of the major collagen genes present in this tissue. It has also been shown that Col II is down regulated in hypothyroid ovarian tissue and its expression is increased upon stimulation by thyroid hormone (T(3)). To know whether less Col II in hypothyroid ovarian tissue is due to less synthesis of the protein or its increased rate of degradation is also involved in it, we demonstrated the status of Collagen - degrading Matrix Metalloproteinases in this condition and found up regulation of MMP-1, -8 and -13 in hypothyroid rat ovary. The present study shows the reduced Col II expression in hypothyroid rat ovary, with the concomitant increase in Col II degradation. This information will be useful for further studies on reproductive disorders.

Tachykinin family genes and their receptors are differentially expressed in the hypothyroid ovary and pituitary.

Plasma tachykinin levels are known to be altered with sexual acyclicity and loss of reproductive function. Ovulatory dysfunction, as seen in postmenopausal women, is also often encountered in hypothyroid patients. To know the involvement of different tachykinin genes in hypothyroidism-associated reproductive disorders, we performed DD-PCR with the pituitary RNA of control and hypothyroid rats to see the differentially expressed gene profile. Subsequently, we selected a few clones, tachykinin being one of them. Since its expression was up regulated in hypothyroidism as it does in the sexually acyclic females, we wanted to correlate these two phenomena with hypothyroidism associated reproductive disorders. We observed differential expression of tac2 along with other tk genes and their receptors in rat pituitary and ovary, which suggests that hypothyroidism affects the expression of these genes in these tissues. The experiments were repeated in ovarian tissue obtained at surgery from hypothyroid human patients, which showed similar expression pattern of TAC3 (equivalent to rat tac2) and their receptors as in rat ovary. Significant reduction of tac2 expression in reproductively less active rat ovary suggests the association of tac2 with reproductive senescence. Our results suggest that decline in reproductive function in hypothyroidism is associated with altered expression level of tac2 and its receptors. Further investigation in this area could elucidate the possible mechanism of tachykinins' involvement in loss of sexual cyclicity and other reproductive disorders associated with hypothyroidism.

Involvement of Pitx2, a homeodomain transcription factor, in hypothyroidism associated reproductive disorders.

Hypothyroid-associated reproductive disorders have now become a striking phenomenon worldwide but the molecular mechanism behind these disorders is not fully known. Pitx2 gene encodes homeodomain transcription factor, which regulates Plod2 gene in brain tissue, transactivates gonadotropin genes in pituitary and plays a substantial role in cell growth and proliferation in different tissues. Pitx2 binds to Plod2 promoter and activates this gene in rat ovary. In this report, we show that Pitx2's expression is markedly reduced in hypothyroid ovary as well as in ovarian granulosa cells, which is recovered with T(3)-supplementation both in vitro and in vivo conditions. Reduced Pitx2 expression could decrease Plod2 expression and hence facilitate ovarian ECM degradation. We have also observed similar pattern of expression of Pitx1 and -3 in hypothyroid and T(3)-supplemented ovaries. The temporal expression of Pitx2 across the estrous cycle shows that it is expressed through all the 4 phases of the cycle and reaches its maximum in the proestrus phase suggesting its possible role in ovulation followed by luteinization. The present study reveals that the reduced Pitx2 expression in hypothyroid ovary could lead to ovarian dysfunction by modulating the Pitx2-Plod2 interaction, further study will be necessary to unravel the complete regulatory mechanism of Pitx2 in ovarian function.