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Quantitative analysis of protein biomarkers in plasma is typically done by ELISA, but this method is limited by the availability of high-quality antibodies. An alternative approach is protein immunoprecipitation combined with multiple reaction monitoring mass spectrometry (IP-MRM). We compared IP-MRM to ELISA for the analysis of six colon cancer biomarker candidates (metalloproteinase inhibitor 1 (TIMP1), cartilage oligomeric matrix protein (COMP), thrombospondin-2 (THBS2), endoglin (ENG), mesothelin (MSLN) and matrix metalloproteinase-9 (MMP9)) in plasma from colon cancer patients and noncancer controls. Proteins were analyzed by multiplex immunoprecipitation from plasma with the ELISA capture antibodies, further purified by SDS-PAGE, digested and analyzed by stable isotope dilution MRM. IP-MRM provided linear responses (r = 0.978-0.995) between 10 and 640 ng/mL for the target proteins spiked into a "mock plasma" matrix consisting of 60 mg/mL bovine serum albumin. Measurement variation (coefficient of variation at the limit of detection) for IP-MRM assays ranged from 2.3 to 19%, which was similar to variation for ELISAs of the same samples. IP-MRM and ELISA measurements for all target proteins except ENG were highly correlated (r = 0.67-0.97). IP-MRM with high-quality capture antibodies thus provides an effective alternative method to ELISA for protein quantitation in biological fluids.
STUDY QUESTION - Can biologically active vitamin D3 [1,25(OH)₂D3] regulate the expression and activity of matrix metalloproteinases (MMPs) in human uterine fibroid cells?
SUMMARY ANSWER - 1,25(OH)₂D3 effectively reduced the expression and activities of MMP-2 and MMP-9 in cultured human uterine fibroid cells.
WHAT IS KNOWN ALREADY - Uterine fibroids (leiomyoma) express higher levels of MMP activity than adjacent normal myometrium, and this is associated with uterine fibroid pathogenesis. However, it is unknown whether 1,25(OH)₂D3 can regulate the expression and activities of MMPs in human uterine fibroid cells.
STUDY DESIGN, SIZE, DURATION - Surgically removed fresh fibroid tissue was used to generate primary uterine fibroid cells.
PARTICIPANTS/MATERIALS, SETTING, METHODS - An immortalized human uterine fibroid cell line (HuLM) and/or primary human uterine fibroid cells isolated from fresh fibroid tissue were used to examine the expression of several MMPs, tissue inhibitors of metalloproteinases (TIMP) 1 and 2 and the activities of MMP-2 and MMP-9 after 1,25(OH)₂D3 treatment. Real-time PCR and western blots analyses were used to measure mRNA and protein expression of MMPs, respectively. Supernatant cell culture media were analyzed for MMP-2 and MMP-9 activities using a gelatin zymography assay.
MAIN RESULTS AND THE ROLE OF CHANCE - 1-1000 nM 1,25(OH)₂D3 significantly reduced mRNA levels of MMP-2 and MMP-9 in HuLM cells in a concentration-dependent manner (P < 0.5 to P < 0.001). The mRNA levels of MMP-1, MMP-3, MMP-13 and MMP-14 in HuLM cells were also reduced by 1,25(OH)₂D3. 1,25(OH)₂D3 significantly reduced MMP-2 and MMP-9 protein levels in a concentration-dependent manner in both HuLM and primary uterine fibroid cells (P < 0.05 to P < 0.001). Moreover, 1,25(OH)₂D3 increased the mRNA levels of vitamin D receptor (VDR) and TIMP-2 in a concentration-dependent manner in HuLM cells (P < 0.05 to P < 0.01). 1,25(OH)₂D3 also significantly increased protein levels of VDR and TIMP-2 in all cell types tested (P < 0.05 to P < 0.001). Gelatin zymography revealed that pro-MMP-2, active MMP-2 and pro-MMP-9 were down-regulated by 1,25(OH)₂D3 in a concentration-dependent manner; however, the active MMP-9 was undetectable.
LIMITATIONS, REASONS FOR CAUTION - This study was performed using in vitro uterine fibroid cell cultures and the results were extrapolated to in vivo situation of uterine fibroids. Moreover, in this study the interaction of vitamin D3 with other regulators such as steroid hormone receptors was not explored.
WIDER IMPLICATIONS OF THE FINDINGS - This study reveals an important biological function of 1,25(OH)₂D3 in the regulation of expression and activities of MMP-2 and MMP-9. Thus, 1,25(OH)₂D3 might be a potential effective, safe non-surgical treatment option for human uterine fibroids.
Angiotensin II (Ang II) is a major contributor to the progression of renal fibrosis. Wang and colleagues provide evidence that signaling through the prolyl-4-hydroxylase domain (PHD)-hypoxia-inducible factor-1 (HIF-1) pathway mediates profibrotic effects of Ang II in rat renal medullary interstitial cells under normoxic conditions, thus placing the HIF oxygen-sensing pathway into the center of an Ang II-induced profibrotic signaling cascade.
BACKGROUND - Cardiac amyloidosis is characterized by amyloid infiltration resulting in extracellular matrix disruption. Amyloid cardiomyopathy due to immunoglobulin light chain protein (AL-CMP) deposition has an accelerated clinical course and a worse prognosis compared with non-light chain cardiac amyloidoses (ie, forms associated with wild-type or mutated transthyretin [TTR]). We therefore tested the hypothesis that determinants of proteolytic activity of the extracellular matrix, the matrix metalloproteinases (MMPs), and their tissue inhibitors (TIMPs) would have distinct patterns and contribute to the pathogenesis of AL-CMP versus TTR-related amyloidosis.
METHODS AND RESULTS - We studied 40 patients with systemic amyloidosis: 10 AL-CMP patients, 20 patients with TTR-associated forms of cardiac amyloidosis, ie, senile systemic amyloidosis (involving wild-type TTR) or mutant TTR, and 10 patients with AL amyloidosis without cardiac involvement. Serum MMP-2 and -9, TIMP-1, -2, and -4, brain natriuretic peptide values, and echocardiography were determined. AL-CMP and TTR-related amyloidosis groups had similar degrees of increased left ventricular wall thickness. However, brain natriuretic peptide, MMP-9, and TIMP-1 levels were distinctly elevated accompanied by marked diastolic dysfunction in the AL-CMP group versus no or minimal increases in the TTR-related amyloidosis group. Brain natriuretic peptide, MMPs, and TIMPs were not correlated with the degree of left ventricular wall thickness but were correlated to each other and to measures of diastolic dysfunction. Immunostaining of human endomyocardial biopsies showed diffuse expression of MMP-9 and TIMP-1 in AL-CMP and limited expression in TTR-related amyloidosis hearts.
CONCLUSIONS - Despite comparable left ventricular wall thickness with TTR-related cardiac amyloidosis, AL-CMP patients have higher brain natriuretic peptide, MMPs, and TIMPs, which correlated with diastolic dysfunction. These findings suggest a relationship between light chains and extracellular matrix proteolytic activation that may play an important role in the functional and clinical manifestations of AL-CMP, distinct from the other non-light chain cardiac amyloidoses.
OBJECTIVE - Hyperhomocysteinemia, neurohormonal activation, inflammation and altered fibrinolysis have been linked to atherothrombosis as well as to myocardial fibrosis and heart failure. Hence, we related a panel of biomarkers representing these pathways to plasma markers of collagen metabolism in a large community-based sample.
METHODS - We related nine biomarkers representing select biologic pathways (independent variables: C-reactive protein, B-type natriuretic peptide, N-terminal proatrial natriuretic peptide, aldosterone, renin, fibrinogen, D-dimer, plasminogen activator inhibitor-1 and homocysteine) to three plasma markers of collagen turnover [dependent variables, separate models for each: aminoterminal propeptide of type III collagen, tissue inhibitor of metalloproteinases-1 and matrix metalloproteinase-9 (present versus absent)] in 921 Framingham Heart study participants (mean age 57 years; 58% women). Participants were separated a priori into those with left ventricular end-diastolic dimensions and wall thickness below sex-specific median values (referent group) and either measure at least 90th sex-specific percentile ('remodeled' group). We used stepwise multivariable regression analysis adjusting for cardiovascular risk factors to relate the panel of systemic biomarkers to the three biomarkers of collagen metabolism.
RESULTS - Plasma homocysteine was positively related to all three markers of collagen metabolism in the remodeled group and to aminoterminal propeptide of type III collagen and tissue inhibitor of metalloproteinases-1 in the referent group. Plasminogen activator inhibitor-1 was positively related to aminoterminal propeptide of type III collagen and tissue inhibitor of metalloproteinases-1 in both groups, whereas the natriuretic peptides were associated positively with these collagen markers in the referent group.
CONCLUSION - In our large community-based sample, plasma homocysteine and plasminogen activator inhibitor-1 were positively related to circulating collagen biomarkers, consistent with experimental studies implicating these pathways in cardiovascular collagen turnover.
BACKGROUND - Aldosterone antagonists reduce morbidity and mortality in patients with severe heart failure, but the mechanisms responsible are not fully understood. Observations in animal models suggest that elevated levels of aldosterone promote oxidative stress and inflammation in the myocardium. It is unknown if these findings are relevant to heart failure patients who may have much lower aldosterone levels.
METHODS AND RESULTS - We therefore examined the relationship of plasma aldosterone levels to markers of oxidative stress, inflammation and matrix turnover in 58 patients with chronic, stable heart failure from systolic dysfunction (LV ejection fraction <0.40) who were not receiving aldosterone antagonists. Chronic, stable heart failure patients had modestly elevated levels of aldosterone. Additionally, these patients had elevated levels of 8-isoprostaglandin F(2alpha), C-reactive protein, soluble intercellular adhesion molecule-1, osteopontin, brain natriuretic peptide, procollagen type III aminoterminal peptide, and tissue inhibitor of metalloproteinase-1. Among these patients with heart failure, aldosterone levels correlated with 8-iso-PGF(2alpha) (P = .003), ICAM-1 (P = .008), and TIMP-1 (P = .006) after adjustment for age, gender, race, diabetes, smoking, heart rate, left ventricular mass, and body mass index.
CONCLUSION - In chronic, stable heart failure patients on standard therapy, higher aldosterone levels are associated with systemic evidence of oxidative stress, inflammation, and matrix turnover.
AIMS - Tissue inhibitor of metalloproteinases-1 (TIMP-1) is a key regulator of extracellular matrix degradation. We examined relations of plasma total TIMP-1 to cardiovascular risk factors and echocardiographic left ventricular (LV) structure and function in a community-based sample.
METHODS AND RESULTS - We studied 1069 Framingham Heart Study participants (mean age 56 years, 58% women) free of heart failure and previous myocardial infarction. Plasma TIMP-1 was higher in men compared with women, and increased with age, body mass index and total/HDL-cholesterol ratio, but decreased with alcohol intake. Plasma TIMP-1 was also directly related to smoking, diabetes and use of anti-hypertensive treatment. Adjusting for age, sex and height, plasma TIMP-1 was positively associated with LV mass, wall thickness, relative wall thickness, end-systolic diameter, and left atrial diameter and the risk of having increased LV end-diastolic diameter or increased wall thickness, and negatively correlated with fractional shortening. Additional adjustment for clinical covariates attenuated the relations of plasma TIMP-1 to most echocardiographic measures.
CONCLUSIONS - In our cross-sectional investigation, plasma total TIMP-1 was related to major cardiovascular risk factors and to indices of LV hypertrophy and systolic dysfunction. This raises the possibility that cardiovascular risk factors may influence cardiovascular remodelling via extracellular matrix degradation, which may be reflected in plasma TIMP-1 levels.
Copyright 2004 Elsevier Ltd
We examined the in vivo function of the angiotensin II type 1 receptor (Agtr1) on macrophages in renal fibrosis. Fourteen days after the induction of unilateral ureteral obstruction (UUO), wild-type mice reconstituted with marrow lacking the Agtr1 gene (Agtr1(-/-)) developed more severe interstitial fibrosis with fewer interstitial macrophages than those in mice reconstituted with Agtr1(+/+) marrow. These differences were not observed at day 5 of UUO. The expression of profibrotic genes - including TGF-beta1, alpha1(I) collagen, and alpha1(III) collagen - was substantially higher in the obstructed kidneys of mice with Agtr1(-/-) marrow than in those with Agtr1(+/+) marrow at day 14 but not at day 5 of UUO. Mice with Agtr1(-/-) marrow were characterized by reduced numbers of peripheral-blood monocytes and macrophage progenitors in bone marrow. In vivo assays revealed a significantly impaired phagocytic capability in Agtr1(-/-) macrophages. In vivo treatment of Agtr1(+/+) mice with losartan reduced phagocytic capability of Agtr1(+/+) macrophages to a level comparable to that of Agtr1(-/-) macrophages. Thus, during urinary tract obstruction, the Agtr1 on bone marrow-derived macrophages functions to preserve the renal parenchymal architecture, and this function depends in part on its modulatory effect on phagocytosis.
Progressive renal disease as a result of renal fibrosis is caused in part by an impairment of the proteolytic machinery that normally regulates matrix turnover. The goal of the present study was to determine whether genetic deficiency of tissue inhibitor of metalloproteinases-1 (TIMP-1) could attenuate interstitial fibrosis caused by unilateral ureteral obstruction (UUO). Groups of wild-type (Timp-1) mice and TIMP-1-deficient (timp-1) mice were killed after 3 and 14 d of UUO or sham operation. Timp-1 mRNA levels were significantly increased 37- and 19-fold in the wild-type mice 3 and 14 d, respectively, after UUO operation. Matrix metalloproteinase-9 (MMP-9) activity fell in all UUO groups but remained significantly higher in the timp-1 group compared with the Timp-1 group. The degree of interstitial fibrosis (kidney collagen content and percentage of tubulointerstitial area stained with picrosirius red and collagen III) was significantly increased 14 d after UUO operation, but there was no difference between the Timp-1 and timp-1 groups. Many features of the fibrogenic response were similar between the Timp-1 and timp-1 groups, including the number of myofibroblasts and the induction of genes encoding procollagen III, fibronectin, and transforming growth factor-beta. After UUO operation, renal mRNA levels for Timp-3 and plasminogen activator inhibitor-1 were significantly higher in the TIMP-1-deficient mice. The results of this study show that elimination of TIMP-1 alone does not alter the severity of interstitial fibrosis. These findings may be due to compensation by other protease inhibitors such as TIMP-2, TIMP-3, and/or plasminogen activator inhibitor-1 or to the possibility that inhibition of intrinsic MMP activity does not constitute a profibrogenic event in the kidney.
BACKGROUND - Progressive renal interstitial fibrosis is characterized by up-regulated expression of the gene that encodes the tissue inhibitor of metalloproteinases-1 (TIMP-1), a regulator of extracellular matrix remodeling, suggesting that impaired matrix turnover contributes to the fibrogenic process. The present study was designed to develop a murine model of renal interstitial fibrosis, and to determine the functional significance of up-regulated Timp-1 expression by comparing the severity of this renal disease in wild-type mice and mice genetically deficient in Timp-1.
METHODS - Initial pilot studies developed and characterized a murine model of bovine serum albumin (BSA)-induced protein-overload proteinuria with respect to the degree of proteinuria, severity of interstitial fibrosis, and renal mRNA levels for genes encoding matrix proteins, transforming growth factor-beta1 (TGF-beta1), and TIMP-1, -2, -3, and -4. In the final study, the severity of interstitial fibrosis was compared in wild-type and Timp-1-deficient mice after six weeks of proteinuria.
RESULTS - Mice injected with large daily intraperitoneal doses of BSA developed proteinuria, interstitial inflammation, and progressive interstitial fibrosis. A time course study based on measurements after one, two, and six weeks of BSA injections showed increased renal mRNA levels for the matrix genes procollagens alpha1(I), alpha1(III), and alpha2(IV) and TGF-beta1 and Timp-1. Timp-2 and Timp-3 genes were constitutively expressed at high levels in the normal kidneys and showed little change in the proteinuric kidneys. Timp-4 transcripts were not detected in any of the kidneys. After six weeks of BSA overload-proteinuria, the groups (N = 8 per group) of wild-type and Timp-1-deficient mice developed significant interstitial fibrosis compared with the control saline-injected groups. The severity of the interstitial fibrosis was similar in both proteinuric groups based on an assessment of the final kidney weight, total kidney collagen content, and the number of interstitial fields with increased fibronectin staining.
CONCLUSIONS - Results of the present study indicate that TIMP-1 deficiency does not alter the degree of interstitial fibrosis in the murine overload proteinuria model. Potential explanations include Timp-1 genetic redundancy, as suggested by the observation that, despite significant intrarenal induction of the Timp-1 gene expression, net renal metalloproteinase-9 (MMP-9) activity was not significantly altered. TIMP-1 is a multifunctional protein that may play a metalloproteinase-independent role in response to renal injury.