Plasma Connective Tissue Growth Factor (CTGF/CCN2)

840 Diabetes Care Volume 41, April 2018

Plasma Connective Tissue Growth Kelly J. Hunt,1,2 Miran A. Jaffa,3 Sara M. Garrett,4 Deirdre K. Luttrell,4 Factor (CTGF/CCN2) Levels Kenneth E. Lipson,5 Maria F. Lopes-Virella,2,4 Predict Myocardial Infarction Louis M. Luttrell,2,4 Ayad A. Jaffa,4,6 and VADT Investigators in the Veterans Affairs Diabetes Trial (VADT) Cohort

Diabetes Care 2018;41:840–846 | https://doi.org/10.2337/dc17-2083

OBJECTIVE Connective tissue growth factor (CTGF), also known as CCN2, is a potent chemotactic and extracellular matrix-inducing matricellular protein that has been implicated in progression of inﬂammatory and ﬁbroproliferative disorders. An emerging role of CTGF/CCN2 is that of a prosclerotic factor implicated in the development of cardiac disease. Our objective was to determine the role of CTGF/CCN2 as a predictor of cardiovascular events in type 2 diabetes in the Veterans Affairs Diabetes Trial (VADT) cohort.

RESEARCH DESIGN AND METHODS Levels of CTGF/CCN2 were measured in 952 VADT patients a median of 1.9 years after entry into the study. Participants were followed for an average of 3.3 years for vascular outcomes. CTGF/CCN2 categories were defined as below the detectable limit (referent, 54.5%), lower half of detectable values (22.8%), and upper half of 1Department of Public Health Sciences, Medical detectable values (22.7%). Hazard ratios (HRs) for cardiovascular end points in re- University of South Carolina, Charleston, SC 2Ralph H. Johnson VA Medical Center, Charles- lation to CTGF/CCN2 categories were calculated by Cox proportional hazards models. ton, SC 3Epidemiology and Population Health Depart- RESULTS ment, Faculty of Health Sciences, American Uni- During follow-up, 4.8% had a myocardial infarction (MI), 6.9% had an MI or cardio- versity of Beirut, Beirut, Lebanon 4 vascular death, and 6.9% died. After adjustments by conventional risk factors, indi- Department of Medicine, Medical University of South Carolina, Charleston, SC viduals in the highest category of CTGF/CCN2 were at higher risk of MI (HR 2.43 [95% 5FibroGen, Inc., San Francisco, CA CI 1.15, 5.14]), MI or cardiovascular death (HR 2.71 [95% CI 1.44, 5.08]), and all-cause 6Department of Biochemistry and Molecular Ge- mortality (HR 2.70 [95% CI 1.43, 5.08]) relative to individuals with CTGF below the netics, Faculty of Medicine, American University of Beirut, Beirut, Lebanon detectable limit. Corresponding author: Kelly J. Hunt, huntke@ CARDIOVASCULAR AND METABOLIC RISK CONCLUSIONS musc.edu, or Ayad A. Jaffa, [email protected]. Our study indicates that high levels of CTGF/CCN2 predict future MI and cardiovas- Received 5 October 2017 and accepted 18 December 2017. cular death in patients with type 2 diabetes. This article contains Supplementary Data online at http://care.diabetesjournals.org/lookup/ Diabetes is associated with a number of metabolic and cardiovascular risk factors that suppl/doi:10.2337/dc17-2083/-/DC1. contribute to a high rate of vascular events. The risk factors and mechanisms that © 2018 by the American Diabetes Association. contribute to the development of these complications are inadequately defined. In the Readers may use this article as long as the work is properly cited, the use is educational and not clinical setting, we strive to maintain glycemic control, promote smoking cessation, and for profit, and the work is not altered. More infor- monitor and treat hypertension and hyperlipidemia to well-established goals. None- mation is available at http://www.diabetesjournals theless, it is clear that our current tools for risk assessment do not provide a complete .org/content/license. care.diabetesjournals.org Hunt and Associates 841

picture, and as a result our treatment (21). It is of interest to note here that substudy focused on determining the as- strategies are suboptimal, particularly CTGF/CCN2 promotes deposition of ex- sociation between specific biomarkers with respect to macrovascular events in tracellular matrix proteins through inter- and macrovascular disease. The bio- patients with established type 2 diabetes. action with other factors. For instance, chemical, physical, and demographic This fact is underscored by recently re- CTGF regulates the fibrosis-associated re- profiles of the 995 patients in the sub- leased outcomes from the CSP-465 Vet- nal lymphangiogensis in obstructed kid- study did not differ significantly from erans Affairs Diabetes Trial (VADT) (1,2), neys via induction and direct interaction those of the 796 not included in the sub- the Action to Control Cardiovascular Risk with vascular endothelial growth factor C study, with the exception of slightly in Diabetes (ACCORD) trial (3), and the (22). In addition, the increased collagen lower age and LDL cholesterol and Action in Diabetes and Vascular Disease: deposition in lung fibrosis as a result of slightly higher triglyceride levels, as Preterax and Diamicron Modified Release deletion of protein phosphatase and ten- well as a higher prevalence of aspirin Controlled Evaluation (ADVANCE) trial sin homologue gene is mediated via in- use at baseline in substudy participants (4), all of which failed to show a significant creased expression of CTGF (23). At the compared with non–substudy partici- benefit of tight glycemic control on car- molecular level, a number of factors have pants (31). The study population for the diovascular events (5). In fact, the inten- been shown to regulate the expression of current report consists of 952 of the sive glycemic control arm of ACCORD was CTGF/CCN2, including advanced glycation 995 participants for whom samples stopped early owing to excess mortality end products, sphingosine-1-phosphate, were available for measurement of (3). The mechanisms responsible for the bradykinin, and LDL (24–29). CTGF. In 43 patients, not enough plasma initiation and progression of atheroscle- In earlier work, we reported that in was collected to perform the measure- rosis are not completely defined. Early type 1 diabetes, higher plasma CTGF/ ments. Supplementary Fig. 1 includes atherosclerotic lesions are characterized CCN2 levels were associated with in- a flow diagram of study participants. by endothelial dysfunction, accumulation creased common and internal carotid Enrollment for the VADT study occurred of inflammatory cells, vascular smooth intima-media thickness, an established from December 2000 to May 2003. Mea- muscle cell proliferation, and transendo- marker of subclinical atherosclerosis, as surement of CTGF was performed on sam- thelial migration, as well as extracellular well as macroalbuminuria and hyperten- ples collected during a routine follow-up matrix deposition in the vessel wall (6). sion (9). In the current study, we build on between July 2002 and March 2006, a Moreover, the accelerated vascular pa- our prior work and examine the ability of median of 1.94 years (range 0–5.03) af- thology associated with diabetes is not CTGF to predict MI in patients with type 2 ter participants’ baseline examination. fully explained by coexistence of tradi- diabetes in the VADT. Plasma samples were obtained after tional cardiovascular risk factors such as an overnight fast and stored at 280°C. hypertension, dyslipidemia, and smoking. RESEARCH DESIGN AND METHODS Patients were followed until lost to follow- Our data implicate a role for connec- VADT Design and Population up, death, or May 2008. The median follow- tive tissue growth factor (CTGF), also The details of the VADT design have pre- up time after measurement of CTGF was known as CCN2, as a mediator of diabetic viously been described (1). Briefly, 1,791 3.76 years (range 0–5.75). CTGF measure- vascular injury. CTGF is a member of the veterans with type 2 diabetes and subop- ment for the current analysis occurred CCN family of matricellular proteins that timal glucose control were randomized in after all samples were collected. The has been implicated in the progression of 20 participating sites to receive either in- baseline VADT cohort examination was the inflammatory process, response to intensive or standard glucose control. The standardized and included interviews, jury, and wound healing (7,8). Dysregula- goal for HbA1c levels was an absolute blood pressure measurements, anthro- tion of CTGF/CCN2 expression has been reduction of 1.5% in the intensive ther- pometric measurements, and fasting linked to various fibroproliferative disor- apy group compared with the standard venipuncture (1). ders including diabetes complications and therapy group. All other modifiable car- atherosclerosis (8,9). An emerging role of diovascular risk factors were treated ag- CTGF/CCN2 Measurement CTGF/CCN2 is that of a prosclerotic factor gressively and uniformly in both arms of CTGF in plasma was measured with a implicated in the development of cardiac the study. All patients were treated ac- sandwich ELISA that detects both intact disease (10–15). CTGF/CCN2 expression cording to guidelines of the American Di- CTGF and CTGF that has been proteoly- is upregulated in cardiac tissue after myo- abetes Association for blood pressure, tically cleaved in the hinge region to re- cardial infarction (MI) and has been hypertension, diet, exercise, and diabetes lease the N-fragment of CTGF (N+W-CTGF shown to modulate cardiac function and education (30). All patients were pre- assay). The capture antibody is human tissue remodeling (16,17). Furthermore, scribed aspirin, and all patients with ele- anti-human CTGF–domain 1 (FibroGen, CTGF/CCN2wasshowntobeaneffector vated lipid levels were prescribed statins Inc., San Francisco, CA). A standard curve of transforming growth factor (TGF)- unless contraindicated. The study was ap- was prepared with rhCTGF (CTGF ex- b–mediated cardiac fibrosis, and the ex- proved by the institutional review board pressed in CHO cells and affinity puri- pression of CTGF in cardiac fibroblasts at each of the participating sites. All pa- fied with an anti-CTGF antibody column; and cardiac myocytes was shown to be tients provided written informed consent. FibroGen). After plate washing, 50 mL mediated via TGF-b (18–20). Moreover, Of the 1,791 VADT study participants, samples, standards, and controls was CTGF/CCN2 has been shown to modulate 995 patients from 17 of the participating added along with 50 mL detection anti- the adverse effects of high glucose and sites, or approximately half from the body (mouse anti-human CTGF–domain free fatty acids on cardiomyocytes func- standard arm and half from the intensive 2 antibody, 500 ng/mL; FibroGen, Inc.) tion such as hypertrophy and apoptosis treatment arm, agreed to participate in a for .1 h. Secondary antibody (100 mL, 842 CTGF/CCN2 and CVD in Type 2 Diabetes Diabetes Care Volume 41, April 2018

goat anti-mouse IgG(H+L)-AP, 1:2,000; intervention for cardiac, cerebrovascular, three groups: those below the detectable Thermo Fisher Scientific, Waltham, MA) or peripheral vascular disease; inoperable limit, ,7.8 ng/mL (54.5%); those with lev- was added for 1 h. Stationary incubation coronary artery disease (CAD); and ampu- els in the lower half of the detectable at 37°C in 100 mL light-sensitive sub- tation for ischemic gangrene. Secondary range, 7.8–13.4 ng/mL (22.8%); and those strate buffer (0.1% diethanolamine in outcomes included MI, CAD, death from with levels in the upper half of the detect- 1.1 mmol/L MgCl2 with 1 mg/mL para- CVD, and death from any cause. CAD in- able range, .13.4–106 ng/mL (22.7%). nitrophenylphosphate substrate) for cluded MI, coronary revascularization Baseline clinical and demographic charac- 5–10 min (depending on yellow color de- procedures, and clinically identified inop- teristics of the cohort are shown in Table velopment) was followed by addition of erable CAD. Cardiovascular death in- 1, stratified by the three categories of 100 mL stop solution (10 N NaOH) to sta- cluded sudden death as defined by the CTGF level. Differences across categories bilize the colored product. Absorbance at Framingham Heart Study, CAD, cerebro- of CTGF were tested using x2 for categor- 405 nm was acquired on a SpectraMax vascular accident, and other cardiovascu- ical characteristics, while means, adjusted 340PC spectrophotometer and analyzed lar events (i.e., cardiomyopathy). for age, ethnic minority status, and treat- with SoftMax Pro 4.8 software (Molecular ment arm, were determined for continu- Devices, Sunnyvale, CA). Statistical Analyses ous variables using linear regression. Prospective analyses were carried out in For cardiovascular time-to-event out- End Points which the plasma level of CTGF, as a bio- comes, Kaplan-Meier survival curves and The primary end point for the VADT was marker, and cardiovascular end points, in- Cox proportional hazards models were the time to the first occurrence of any one cluding MI and the composite end point, used to calculated hazard ratios (HRs) for of a composite of cardiovascular events. were the outcomes of interest. Since the end points of interest in relation to CTGF Each VADT event was adjudicated by an CTGF assay was designed to measure lev- categories. Because CTGF levels were mea- end point committee that used strict al- els above median values, slightly more sured not at the baseline VADT examina- gorithms to define and document each than half (54.5%) of VADT participants tion but, instead, a median of 1.94 years event. The composite end point included had CTGF levels below the detectable later, left truncation was used to account documented MI; stroke; death from car- level; therefore, for purposes of statistical for differences in time at risk; hence, a par- diovascular disease (CVD); new or wors- analyses, individuals were categorized ticipant was considered at risk for a given ening congestive heart failure; surgical based on their CTGF level into one of event between measurement of CTGF

Table 1—Clinical and demographic characteristics of the VADT population (n = 952) stratiﬁed by CTGF category CTGF categories (cut points), ng/mL Total Below detectable population limit ,7.8 (n = 519) $7.8–13.4 (n = 217) .13.4–106 (n = 216) P** Measured at VADT baseline Age (years)* 59.7 (59.2, 60.3) 57.9 (57.2, 58.6) 61.1 (60.0, 62.2) 62.7 (61.6, 63.8) ,0.0001 Diabetes duration (years) 11.4 (10.9, 11.9) 10.5 (9.9, 11.1) 11.5 (10.5, 12.5) 13.5 (12.5, 14.5) ,0.0001 Male* 97.1 95.95 98.62 98.15 0.0835 Non-Hispanic white* 60.0 54.91 66.82 65.28 0.0021 Intensive treatment* 49.7 46.63 53.46 54.17 0.0866 Prior vascular event* 38.5 33.53 39.63 49.07 0.0004 Current smoker* 17.3 19.85 12.56 15.74 0.0472 Hypertension* 73.1 69.19 76.96 78.70 0.0105 Exercise* 44.5 43.91 45.83 44.44 0.8918 Adherence to diet* 46.7 45.95 47.69 47.69 0.8667 Aspirin* 91.8 92.26 90.74 91.63 0.7885 Measured at time of CTGF measurement Statin* 79.0 78.72 77.31 81.40 0.5662 ACE* 68.5 68.09 67.59 70.23 0.8099 ARB* 12.6 10.83 15.74 13.49 0.1681

HbA1c (%) 8.0 (7.9, 8.1) 8.0 (7.9, 8.1) 7.9 (7.8, 8.1) 8.1 (7.9, 8.2) 0.6426 ACR‡ 19 (17, 21) 13 (11, 15) 23 (18, 29) 38 (30, 48) ,0.0001 eGFR 79.6 (78.1, 81.1) 84 (83, 86) 77 (74, 80) 70 (67, 73) ,0.0001 BMI (kg/m2) 32.4 (32.0, 32.7) 31.7 (31.3, 32.2) 32.9 (32.2, 33.5) 33.4 (32.7, 34.0) ,0.0001 SB pressure (mmHg) 127 (126, 128) 126 (125, 128) 128 (125, 130) 130 (128, 132) 0.0244 DB pressure (mmHg) 72.8 (72, 73) 73 (72, 74) 72 (71, 73) 73 (71, 74) 0.3449 HDL cholesterol (mg/dL) 38 (37, 39) 39 (38, 40) 37 (35, 38) 37 (36, 39) 0.0547 LDL cholesterol (mg/dL) 96 (94, 98) 95 (92, 98) 94 (89, 98) 100 (96, 105) 0.0487 Triglycerides (mg/dL)‡ 153 (148, 159) 145 (138, 153) 156 (145, 168) 172 (160, 186) 0.0012 Continuous characteristics are shown as means with associated 95% CIs, while categorical characteristics are shown as percentages. Characteristics of the study population adjusted for age, minority status, and treatment arm of the study. ACR, albumin-to-creatinine ratio; ARB, angiotensin II receptor blockers; DB, diastolic blood; SB, systolic blood. *Unadjusted. ‡Owing to nonnormal distributions, geometric means are presented. **x2 for categorical variables and F test for continuous variables. care.diabetesjournals.org Hunt and Associates 843

levels and the end of VADT follow-up. Ini- MI or cardiovascular death after mea- Table 2—VADT cardiovascular events tial models examining the association be- surement of CTGF (Table 2). after measurement of CTGF (total tween the three CTGF categories and each Figure 1 depicts Kaplan-Meier survival N =952) cardiovascular event of interest were as- curves for MI (Fig. 1A), MI combined with Cardiovascular end points N (%) sessed after age, ethnic minority, and cardiovascular death (Fig. 1B), and all- MI 46 (4.83) treatment arm were controlled for. Sec- cause mortality (Fig. 1C). Cox proportional MI, procedure or inoperable 105 (11.03) ondary models additionally adjusted for hazards models were used to examine the MI or cardiovascular death 66 (6.93) prior cardiovascular event, hypertension ability of CTGF to predict VADT macrovas- Composite end point* 166 (17.44) status, and smoking status at date of ran- cular end points (Table 3). After adjust- Death 66 (6.93) domization, as well as LDL cholesterol ment for age, minority status, treatment *Composite end point includes documented level, triglycerides, use of ACE inhibitors, arm, prior cardiovascular event, hyper- MI; stroke; death from cardiovascular causes; and use of statins at time of CTGF mea- tension status and smoking status at new or worsening congestive heart failure; surement. The final model also adjusted date of randomization, as well as LDL cho- surgical intervention for cardiac, fi cerebrovascular, or peripheral vascular for estimated glomerular ltration rate lesterol level, triglycerides, use of ACE disease; inoperable CAD; and amputation for (eGFR) at time of CTGF measurement. inhibitors, and use of statins at time of ischemic gangrene. These covariates were chosen a priori, CTGF measurement, individuals with since they represent either study design CTGF level .13.4 ng/mL, compared with variables or established cardiovascular individuals with CTGF level ,7.8 ng/mL, risk factors. had significantly higher risk of MI (HR 2.36 with procedure or inoperable disease Appropriate interaction terms were [95% CI 1.16, 4.84]), MI or cardiovascular (i.e., not limited to acute events), the HR used to determine whether treatment death (HR 2.76 [95% CI 1.51, 5.04]), and declined and was not significant, indicat- arm, ethnic minority status, or prior CVD death from any cause (HR 3.02 [95% CI ing that CTGF may be related to acute event modified the relationship between 1.66, 5.50]). This significantly increased events through a mechanism other than CTGF categories and outcomes of in- risk was not observed in comparison of its association with atherosclerotic bur- terest. Potential effect modifiers were individuals with CTGF level between 7.8 den, for which CTGF has been postulated chosen a priori, since they represent ei- and 13.4 ng/mL with individuals with to be a plaque-stabilizing factor (12). ther study design variables or established CTGF level ,7.8 ng/mL (Table 3). More- A major difference between the VADT cardiovascular risk factors. The assump- over, additional adjustment for eGFR at and DCCT/EDIC (Diabetes Control and tion of proportional hazards was evalu- time of the biomarker measurement Complications Trial/Epidemiology of ated by testing for interaction between had only a minimal impact on the HR for Diabetes Interventions and Complica- the two CTGF index variables (i.e., defin- MI (HR 2.43 [95% CI 1.15, 5.14]) and MI or tions) cohorts besides diabetes type (i.e., ing the three CTGF categories) and con- cardiovascular death (HR 2.71 [95% CI type 1 vs. type 2 diabetes) is the level of tinuous time variables. Reported P values 1.44, 5.08]) and a slightly larger impact CVD at the time of measurement of CTGF. are two sided with a type I error rate sig- on the HR for all-cause mortality (HR 2.70 Patients enrolled in the VADT study were nificance level of a = 0.05. HRs (95% CI) are [95% CI 1.43, 5.08]). There was no evidence older, had a longer duration of diabe- displayed. All analyses were performed us- that study treatment arm, minority status, tes and a high comorbidity burden, and ing SAS, version 9.4 (SAS Institute, Cary, NC). or prior history of a cardiovascular event were likely to have a history of CVD (i.e., modified the association between CTGF 38.5%). DCCT/EDIC participants, on the RESULTS level and any of the macrovascular end other hand, were younger, had a shorter At VADT baseline, the mean age of the points examined. duration of diabetes and a low comorbid- study population was 59.7 years and the ity burden, and had not had a cardiovas- mean duration of diabetes was 11.4 CONCLUSIONS cular event prior to enrollment. Results years. Of the 952 participants studied, We previously reported that in type 1 di- from both the DCCT/EDIC and VADT, 97.1% were male, 60% were non-Hispanic abetes, increased plasma CTGF levels which collectively include participants white, 38.5% had experienced a prior were associated with increased common across the continuum of diabetes disease event, and 49.7% were assigned to the and internal carotid intima-media thick- burden, indicate that CTGF may play a role VADT intensive treatment group. HbA1c ness, an established marker of subclinical not only in development of atherosclerosis levels, diastolic blood pressure, and HDL atherosclerosis, as well as macroalbumi- butalsoinpredictionofacuteeventsand, cholesterol remained similar across the nuria and hypertension (9). In the current as such, may have substantial value both three categories of CTGF after adjustment study, we report CTGF is a strong predic- as a biological marker of inflammation- for age, ethnic minority status, and treat- tor of MI, MI combined with cardiovascu- induced tissue injury and as a therapeu- ment arm of the study. Also, there was no lar death, and all-cause mortality. The tic target. association of CTGF categories with treat- ability of CTGF to predict these end points The findings of the current study also ment for statins, ACE inhibitors, or aspirin. remained after established cardiovascu- point to a relationship between plasma BMI, systolic blood pressure, albumin-to- lar risk factors were controlled for, includ- CTGF levels and renal function. Our data creatinine ratio, eGFR, LDL cholesterol, ing age, minority status, VADT treatment indicated that the mean eGFR levels de- and triglycerides were statistically signifi- arm, history of CVD, history of hyperten- clined across increasing categories of cantly different among the three CTGF sion, lipid levels, smoking status, use of CTGF. This is in line with our previous groups (Table 1). During the follow-up pe- ACE inhibitors, use of statins, and eGFR. findings in which increased levels of riod, 46 had an MI and 66 experienced an Moreover, when incident MI was combined plasma CTGF N-fragment were associated 844 CTGF/CCN2 and CVD in Type 2 Diabetes Diabetes Care Volume 41, April 2018

result of increased production (9). It is important to point out that adjustment for eGFR had little impact on the HRs of CTGF between model 2 and model 3 (Table 3), indicating that renal function is not a confounder of the relationship between CTGF and outcomes of interests (i.e., MI, MI and cardiovascular death, all- cause mortality). The increased risk of cardiovascular events seen in patients with type 2 diabetes is associated with a cluster of risk factors for cardiovascular and metabolic disorders that tend to coexist in these patients, including central adiposity, hypertension, and dyslipidemia (32). How- ever, the complex signaling networks through which diabetes, hypertension, and dyslipidemia accelerate vascular damage and trigger acute vascular events are not well-defined. The modifiable factors engaged in these processes have yet to be identified, but there is evidence for promotion of chronic low-grade inflammation, oxidative stress, endothelial dysfunction, stimulation of proliferative/ apoptotic pathways, and deposition of extracellular matrix. Importantly, inflammatory mediators and growth factors are increasingly recognized as key players in the pathogenesis of macrovascular disease (33). Emerging studies indicate that CTGF/ CCN2 is a pathogenic risk determinant acting along a causal pathway for development of CVD. CTGF is a secreted proad- hesive matricellular protein that has been implicated in modulating inflammatory and fibroproliferative disorders (34). The expression of CTGF mRNA and protein levels were increased in vascular cells of advanced atherosclerotic lesions and were found to be aggregated predo- minantly in areas near the fibrous cap of the plaque, suggesting potential involve- ment in atherosclerotic plaque production (35,36). In addition, inhibition of CTGF in the cardiovascular system was shown to reverse tissue remodeling and the process of fibrosis (37). More- over, plasma CTGF was linked to an increased risk of cardiovascular events and mortality in patients with atheroscle- Figure 1—Kaplan-Meier survival curves for incident MI (A), MI combined with cardiovascular death (B), and all-cause mortality (C). *The time axis starts at time of randomization, and left truncation is rotic disease and was associated with pla- used to account for differences in time at risk, since CTGF was measured on samples collected que stabilization after stroke (12,15). a median of 1.94 years after randomization. Med, medium. Although the exact mechanisms through which CTGF mediates its inflammatory with decreased eGFR in patients with with type 1 diabetes with overt albumin- and fibrotic effects on the vasculature type 1 diabetes (9). The increase in uria was not the result of a decrease in are not fully defined, engagement of all plasma CTGF levels observed in patients renal clearance of CTGF but, rather, was a six members of integrin receptors and care.diabetesjournals.org Hunt and Associates 845

Table 3—Adjusted HRs (95% CI) from Cox proportional hazards regression models of end points, with 46 events for MI and for CTGF in relation to various outcomes* in the total population 66 events when MI is combined with car- CTGF diovascular death. In summary, this study demonstrates Model 1** Model 2† Model 3‡ for the first time that high levels of MI CTGF/CCN2 predict future MI and cardio- Below detectable limit 1.00 1.00 1.00 vascular death in patients with type 2 di- 7.8–13.4 ng/mL 1.28 (0.59, 2.75) 1.34 (0.61, 2.95) 1.36 (0.62, 3.02) .13.4 ng/mL 2.24 (1.13, 4.45) 2.36 (1.16, 4.84) 2.43 (1.15, 5.14) abetes. Plasma CTGF/CCN2 levels warrant further study as a potential biomarker to MI, procedure or inoperable disease fi Below detectable limit 1.00 1.00 1.00 be used for risk strati cation with respect 7.8–13.4 ng/mL 0.77 (0.45, 1.31) 0.79 (0.46, 1.36) 0.79 (0.46, 1.37) to prediction of cardiovascular events. .13.4 ng/mL 1.40 (0.88, 2.23) 1.32 (0.82, 2.13) 1.32 (0.80, 2.17) Moreover, further understanding of the MI or cardiovascular death mechanisms responsible for the reported Below detectable limit 1.00 1.00 1.00 relationship between CTGF/CCN2 and 7.8–13.4 ng/mL 1.78 (0.95, 3.33) 1.63 (0.84, 3.18) 1.62 (0.83, 3.17) acute vascular events may identify novel . 13.4 ng/mL 2.79 (1.55, 5.01) 2.76 (1.51, 5.04) 2.71 (1.44, 5.08) targets for therapeutic interventions. Composite end point Below detectable limit 1.00 1.00 1.00 7.8–13.4 ng/mL 1.02 (0.69, 1.53) 0.94 (0.62, 1.42) 0.93 (0.61, 1.42) .13.4 ng/mL 1.61 (1.11, 2.33) 1.44 (0.99, 2.10) 1.43 (0.97, 2.11) Acknowledgments. The authors thank the VADT Executive Committee for review and ap- All death proval of the manuscript for submission. Below detectable limit 1.00 1.00 1.00 Funding. This work was supported by the Na- 7.8–13.4 ng/mL 1.76 (0.90, 3.46) 1.39 (0.68, 2.87) 1.30 (0.62, 2.71) tional Institutes of Health National Heart, Lung, .13.4 ng/mL 3.30 (1.82, 5.99) 3.02 (1.66, 5.50) 2.70 (1.43, 5.08) and Blood Institute grants RO1-HL-077192 and *The number of events for each outcome for each model are as follows: MI, 46, 45, and 45 for model PO1-HL-55782 and the Department of Veterans 1, model 2, and model 3, respectively; MI, procedure or inoperable disease, 105, 104, and 104; MI Affairs. or cardiovascular death, 66, 63, and 63; composite end point, 166, 162, and 162; and all death, 66, The funding sources of this study did not play 63, and 63. **Adjusted for age, minority, and treatment arm. †Additionally adjusted for prior a role in the study design; in the collection, analysis, cardiovascular event, hypertension status, and smoking status at date of randomization, as well as or interpretationof data; in thewritingofthereport; LDL, triglycerides, use of ACE inhibitors, and statin use at time of CTGF measurement. ‡Additionally or in the decision to submit the manuscript for adjusted for eGFR at time of CTGF measurement. publication. The content of this article does not represent the views of the Department of Veterans Affairs or the U.S. government. Duality of Interest. K.E.L. reports personal fees binding to heparin sulfate proteoglycans across a continuum of levels. Second, be- and other from FibroGen, Inc., outside the sub- such as syndecan-4, LDL-related receptor cause the vascular disease burden was mitted work. No other potential conflicts of inter- protein, and the cation-independent high among patients at enrollment into est relevant to this article were reported. mannose-6-phosphate receptor have the VADT study, our measurement of Author Contributions. K.J.H. analyzed data and wrote, reviewed, and edited the manuscript. been implicated as key factors through CTGF, while occurring prior to develop- M.A.J. reviewed the analytical plan and edited which CTGF transduces its cellular effects ment of acute cardiovascular events, the manuscript. S.M.G. and D.K.L. oversaw the (38). It is of interest to point out here that likely represents CTGF measured when measurements of plasma CTGF and reviewed CCN3 (nov), another member of the CCN atherosclerotic burden was high. Hence, and edited the manuscript. K.E.L., M.F.L.-V., and family, has been shown to act down- it is difficult to determine the temporal L.M.L. reviewed and edited the manuscript. A.A.J. b conceived the study, amassed the resulting data, stream of TGF- to inhibit the expression relationship between high plasma CTGF and wrote, reviewed, and edited the manuscript. of CCN2 and, hence, attenuate deposition levels and development of atherosclero- K.J.H. and A.A.J. had final responsibility for the of extracellular matrix proteins (39,40). sis and whether CTGF levels were ele- decision to submit the manuscript for publication. CCN3 functions as an endogenous nega- vated because of the high burden of A.A.J. was the underwriter of the study. K.J.H. and tive modulator of the profibrotic actions atherosclerosis or whether they played A.A.J. are the guarantors of this work and, as such, had full access to all the data in the study and take of CCN2 in vivo to promote deposition of a causal role in the development of ath- responsibility for the integrity of the data and the matrix proteins, thus providing a mecha- erosclerosis. It is also difficult to know accuracy of the data analysis. nistic pathway in which the activity of whether the association between CTGF Prior Presentation. Parts of this study were CCN2 can be regulated in disorders of and acute events only reflects an associ- presented in abstract form at the 77th Scientific fi Sessions of the American Diabetes Association, brosis (39,40). ation between high atherosclerotic bur- San Diego, CA, 9–13 June 2017. One limitation of our study is that den and increased risk of acute events the assay used to measure CTGF was or whether CTGF levels could also be re- References designed to measure CTGF levels above lated mechanistically to acute events. Our 1. Abraira C, Duckworth W, McCarren M, et al.; median values in the population; hence, analysis, which reports an association VA Cooperative Study of Glycemic Control and slightly more than half of the VATD pop- with MI and MI combined with cardiovas- Complications in Diabetes Mellitus Type 2. Design ulation had CTGF levels below the detect- cular death, but not with MI combined of the cooperative study on glycemic control and able limit of the assay. Thus, we were with procedure or inoperable disease, complications in diabetes mellitus type 2: Veter- ans Affairs Diabetes Trial. J Diabetes Complica- forced to group all individuals below the suggests that CTGF levels may be related tions 2003;17:314–322 detectable limit into a single category and mechanistically to acute events. A third 2. Duckworth W, Abraira C, Moritz T, et al.; VADT were therefore unable to examine CTGF limitation is the somewhat low number Investigators. Glucose control and vascular 846 CTGF/CCN2 and CVD in Type 2 Diabetes Diabetes Care Volume 41, April 2018

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