August 2014
Volume 55, Issue 8
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Glaucoma  |   August 2014
Elevated Transforming Growth Factor β1 in Plasma of Primary Open-Angle Glaucoma Patients
Author Notes
  • Vanderbilt Eye Institute, Vanderbilt University, Nashville, Tennessee, United States 
  • Correspondence: Rachel W. Kuchtey, Vanderbilt Eye Institute, Vanderbilt University, 2311 Pierce Avenue, Nashville, TN 37232, USA; rachel.kuchtey@vanderbilt.edu
Investigative Ophthalmology & Visual Science August 2014, Vol.55, 5291-5297. doi:10.1167/iovs.14-14578
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      John Kuchtey, Jessica Kunkel, L. Goodwin Burgess, Megan B. Parks, Milam A. Brantley, Rachel W. Kuchtey; Elevated Transforming Growth Factor β1 in Plasma of Primary Open-Angle Glaucoma Patients. Invest. Ophthalmol. Vis. Sci. 2014;55(8):5291-5297. doi: 10.1167/iovs.14-14578.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose.: To test the hypothesis that primary open-angle glaucoma (POAG) patients have a systemic elevation of transforming growth factor β1 (TGFβ1).

Methods.: Plasma was prepared from blood samples drawn from patients of the Vanderbilt Eye Institute during clinic visits. Concentrations of total TGFβ1 and thrombospondin-1 (TSP1) in plasma were determined by ELISA. Statistical significance of differences between POAG and control samples was evaluated by Mann-Whitney test. Regression analysis was used to evaluate correlations between plasma TGFβ1 and patient age and between plasma TGFβ1 and TSP1.

Results.: Plasma samples were obtained from 148 POAG patients and 150 controls. Concentration of total TGFβ1 in the plasma of POAG patients (median = 3.25 ng/mL) was significantly higher (P < 0.0001) than in controls (median = 2.46 ng/mL). Plasma TGFβ1 was not correlated with age of patient (P = 0.17). Thrombospondin-1 concentration was also significantly higher (P < 0.0001) in POAG patients (median = 0.774 μg/mL) as compared to controls (median = 0.567 μg/mL). Plasma total TGFβ1 and TSP1 concentrations were linearly correlated (P < 0.0001).

Conclusions.: Plasma samples from POAG patients display elevated total TGFβ1 compared to controls, consistent with elevated systemic TGFβ1 in POAG patients.

Introduction
Transforming growth factor βs (TGFβs) are multifunctional cytokines that play central roles in a wide variety of physiological responses such as regulation of immune responses, cell proliferation and differentiation, vascular homeostasis, wound healing, and fibrosis. 14 Depending on the specific microenvironment in which they act, TGFβs can exert opposing effects, 5 for example, promoting or inhibiting immune reactions or cell proliferation. Mammals express three isoforms of TGFβ encoded by separate genes: TGFβ1, TGFβ2, and TGFβ3. Transforming growth factor βs are secreted in a latent form that consists of dimers of latent TGFβ bound to latent TGFβ binding protein (LTBP). 6 Transforming growth factor β2 is the predominant isoform in aqueous humor (AH), 7,8 while TGFβ1 is the predominant isoform in plasma. 911 In the AH, TGFβ2 plays a central role in anterior chamber-associated immune deviation. 12  
Elevation of TGFβ2 in the AH of patients with primary open-angle glaucoma (POAG) was first reported in 1994 by Tripathi et al. 13 A series of published studies from several laboratories followed, 1420 reporting consistent findings as reviewed by Prendes et al., 21 leading to a widely held consensus that TGFβ2 tends to be elevated in the AH of glaucoma patients. Transforming growth factor β1 has also been shown to be elevated in AH from POAG patients, though its concentration is ∼40-fold lower than that of TGFβ2 in normal AH. 17,22,23  
As recently reviewed by Fuchshofer and Tamm, 24 a pathogenic role for elevated TGFβ2 in glaucoma is supported by multiple studies showing, for example, that perfusion of human anterior segments with TGFβ2 results in decreased outflow facility, 25,26 and overexpression of TGFβ2 in rodent eyes leads to glaucoma phenotypes. 2729 Transforming growth factor β2 overexpression is hypothesized to promote glaucoma through effects on extracellular matrix turnover, affecting both AH outflow facility 2426 and susceptibility of the optic nerve to pressure-induced damage. 30  
Microfibrils are a major reservoir of latent TGFβs. All three TGFβ isoforms can associate with microfibrils through interactions between LTBP and fibrillin-1, the principal component of microfibrils. 3134 Microfibrils are located in the extracellular matrix of a variety of tissues where they play a central role in localization and activation of TGFβ signaling. 3134 In the eye, microfibrils are found in the trabecular meshwork, inner wall of Schlemm's canal, iris, ciliary body, and optic nerve head. 3537 In addition to microfibrils, platelets are another major reservoir of latent TGFβ1 that is released upon platelet degranulation. 38  
Elevated systemic TGFβ1 levels and chronic activation of TGFβ signaling are associated with a number of diseases caused by mutations in the fibrillin-1 gene (FBN1) and in other microfibril-associated genes. 3949 Microfibril defects may also cause glaucoma, 50 as suggested by the association of the microfibril gene LTBP2 5153 with primary congenital glaucoma in humans. Recently, we identified a mutation in a microfibril-related gene, ADAMTS10, as causative for POAG in dogs. 54,55 Association of microfibril-related genes and glaucoma has led us to form the hypothesis that microfibril defects cause glaucoma. 50,55 Our microfibril hypothesis of glaucoma would predict that glaucoma patients may have systemic elevation of TGFβ1, similar to observations in other microfibril-related diseases. To investigate this possibility, we have compared TGFβ1 concentrations in plasma samples from POAG patients and control subjects in the present study. 
Methods
Study Participants
This study was approved by the institutional review board of Vanderbilt University and followed the tenets of the Declaration of Helsinki. Written informed consent was obtained from all subjects after explanation of the nature and possible consequences of the study. All participants were patients of the glaucoma or retina services of the Vanderbilt Eye Institute. Clinical diagnosis of POAG was made by a fellowship-trained glaucoma specialist (RWK) based on optic nerve appearance characteristic of glaucoma, such as progressive cupping, thinning of the neuroretinal rim, notch formation, and disc hemorrhage. All patients underwent ancillary tests indicated by their clinical findings, such as visual field test (Humphrey visual field analyzer; Carl Zeiss Meditec, Dublin, CA, USA), fundus photographs of optic nerve (Zeiss 450+ fundus camera; Carl Zeiss Meditec), and spectral-domain optical coherence tomography (Cirrus OCT; Carl Zeiss Meditec). Elevated intraocular pressure was not used as a diagnostic criterion. All patients had open iridocorneal angles as determined by gonioscopy examination. Control patients were >50 years of age and were examined by ophthalmologists and found to be not affected by glaucoma. Patients were excluded from the study if they had been diagnosed with any form of glaucoma other than POAG, optic nerve degeneration not due to glaucoma, age-related macular degeneration, diabetic retinopathy, retinal vascular diseases, or an inherited retinopathy. Additional exclusion criteria were history of ocular inflammation or infection, surgery within the past 2 months, cancer, or renal failure. Family history of glaucoma was determined by patient interview. Current medications were determined by examining patient medical records. 
Blood Collection and Plasma Preparation
Approximately 5 mL blood was collected from patients by venipuncture using a 23-gauge needle connected via tubing to an evacuated tube coated with lithium heparin (BD Vacutainer, catalog no. 366667; BD, Franklin Lakes, NJ, USA). Prior to blood draw, a tourniquet was applied to facilitate locating the cubital vein. The tourniquet was removed upon initiation of blood draw. Blood samples were kept on ice prior to plasma preparation. To prepare plasma, blood was centrifuged at 4°C for 10 minutes at 1500g in a 45° fixed-angle rotor within 2 hours of collection. Since platelets are a major source of TGFβ1 in blood, 9,38 initial studies were performed to evaluate the effectiveness of a single centrifugation step in fully removing platelets from the plasma. Addition of a second centrifugation did not reduce plasma TGFβ1 concentration (P = 0.86), indicating that a single centrifugation step was sufficient to remove platelets (data not shown). Therefore, a single centrifugation step as described above was used to prepare plasma for all samples in this study. Immediately after centrifugation, plasma samples were aliquoted and stored in a −80°C freezer located in the eye clinic. All blood collection and plasma preparation was carried out by two dedicated study coordinators (LGB and MBP) using consistent technique at a single location (Vanderbilt Eye Institute). 
ELISA Assays for TGFβ and Thrombospondin-1
Plasma concentrations of thrombospondin-1 (TSP1) and total (active plus latent) TGFβ1 and total TGFβ2 were determined using well-established 5658 commercially available sandwich design ELISA kits (DuoSet ELISA; R&D Systems, Minneapolis, MN, USA), following the manufacturer's protocols including suggested sample diluents. Transforming growth factor β was converted to its active form by addition of 6 μL 1 N HCl to 12 μL plasma followed by 10-minute incubation at room temperature, neutralization by addition of 6 μL 1.2 N NaOH/0.5 M HEPES, and addition of 216 μL sample diluent. For TSP1 ELISA, plasma samples were diluted 1:11 in sample diluent. Duplicate wells were included for all samples. Each ELISA plate included approximately equal numbers of samples from control and POAG patients. Serial dilutions of recombinant protein standards were included in each plate to generate calibration curves. 
Optical density of plates at 450 nm (OD450) and 540 nm (OD540) were determined with a SpectraMax M5 microplate reader (Molecular Devices, Sunnyvale, CA, USA). Correction for optical imperfections in the plates was made by subtracting OD540 from OD450. A calibration curve of OD450 − OD540 readings versus known concentration of the standards was calculated using a four-parameter logistic fitting routine included in the microplate software (SoftMax Pro, v.5.3.1; Molecular Devices). The working detection range of the ELISAs was approximately 0.62 to 40 ng/mL for TGFβs and 0.18 to 2.2 μg/mL for TSP1, based on the concentration range of calibration samples and dilution of the plasma samples. 
Platelet Activation
The potent platelet-activating peptide TRAP6, which corresponds to amino acid residues 42 to 47 of the thrombin receptor (SFLLRN), 59 was added to 1-mL aliquots of blood for final concentrations of 0, 0.1, 1, and 10 μM. Plasma was prepared as above after 5-minute incubation at room temperature. 
Statistics
Differences in TGFβ1 or TSP1 concentrations between POAG and controls were evaluated by two-tailed Mann-Whitney tests using the online resource VassarStats (http://vassarstats.net/ [in the public domain]), with P values < 0.025 considered significant, taking multiple testing into consideration. Significance of correlations was evaluated by correlation and regression analysis using VassarStats. Comparisons between slopes of best-fit lines were made by analysis of covariance (ANCOVA) using GraphPad Prism software, version 6.04 (GraphPad Software, La Jolla, CA, USA). Significance of differences between control and POAG patient characteristics shown in the Table was examined by Student's t-test for age and by Fisher's exact test for all other patient characteristics using an online resource (http://www.langsrud.com/stat/index.html [in the public domain]). 
Table
 
Patient Characteristics
Table
 
Patient Characteristics
Control POAG P Value*
Number of patients 150 148
Age, y, mean (SD) 66.3 (7.8) 66.2 (12.2) 0.94
Female, n (%) 82 (54.7) 89 (60.1) 0.35
Caucasian, n (%) 143 (95.3) 136 (91.9) 0.25
African American, n (%) 6 (4.0) 9 (6.1) 0.44
Asian, n (%) 1 (0.7) 3 (2.0) 0.37
Primary family history,† n (%) 12 (8.0) 41 (27.7) 8.00 × 10−6
Any family history,‡ n (%) 19 (12.7) 74 (50.0) 5.04 × 10−6
Results
Plasma samples were obtained from 148 POAG patients and 150 control patients. The two groups did not significantly differ in age, sex, or ancestry (Table). Primary open-angle glaucoma patients reported family history of glaucoma at a significantly higher rate than control patients (P < 10−5, Table), consistent with previous studies. 60,61  
In an initial experiment comparing plasma samples from 10 control and 10 POAG patients, total TGFβ1 was readily detectable while total TGFβ2 was below limits of detection for all samples (data not shown). This relative abundance of TGFβ1 compared to TGFβ2 was consistent with previous studies showing TGFβ1 highly expressed in plasma, with low levels or no expression of TGFβ2. 911,62 Active TGFβ1, determined with plasma samples not activated by acid treatment, was also below detection limits. Based on these initial findings, the present study focused on the total amount of the TGFβ1 isoform. 
Total TGFβ1 was elevated in plasma samples from POAG patients as compared to controls (Fig. 1). Median TGFβ1 concentration for POAG was 3.25 ng/mL, with an intraquartile range of 2.4 to 4.9 ng/mL, compared to a median of 2.46 ng/mL and intraquartile range of 1.8 to 3.6 ng/mL for control plasma samples. While there was considerable variability between individuals and overlap between groups, the difference was significant (P < 0.0001). Plasma TGFβ1 was not linearly correlated with age (Fig. 2) for either the control (P = 0.17) or POAG samples (P = 0.79), consistent with previous studies. 9,39  
Figure 1
 
Total TGFβ1 concentration in the plasma of control and POAG patients. TGFβ1 is significantly higher in the POAG group (P < 0.0001). Box plots are shown superimposed on individual data points (blue circles) with the lower and upper horizontal lines of the boxes representing the first and third quartiles, respectively, and the extent of the vertical lines above and below the boxes indicating the 90th and 10th percentiles, respectively.
Figure 1
 
Total TGFβ1 concentration in the plasma of control and POAG patients. TGFβ1 is significantly higher in the POAG group (P < 0.0001). Box plots are shown superimposed on individual data points (blue circles) with the lower and upper horizontal lines of the boxes representing the first and third quartiles, respectively, and the extent of the vertical lines above and below the boxes indicating the 90th and 10th percentiles, respectively.
Figure 2
 
Total TGFβ1 concentration in the plasma is not correlated with patient age for either controls ([A], P = 0.17) or POAG patients ([B], P = 0.79). Plasma total TGFβ1 concentration was plotted versus age at time of blood draw. Equation of the best-fit line and R 2 value are shown in the upper left corner of each plot.
Figure 2
 
Total TGFβ1 concentration in the plasma is not correlated with patient age for either controls ([A], P = 0.17) or POAG patients ([B], P = 0.79). Plasma total TGFβ1 concentration was plotted versus age at time of blood draw. Equation of the best-fit line and R 2 value are shown in the upper left corner of each plot.
Since platelets release abundant amounts of both TGFβ1 and TSP1 upon activation, plasma TSP1 concentration was measured as a marker of platelet degranulation in the same set of samples as used for TGFβ1 determinations. Similar to TGFβ1, TSP1 was elevated in POAG plasma compared to controls (P < 0.0001), with similar variability and overlap in distributions (Fig. 3). Median TSP1 concentration for POAG was 0.774 μg/mL with intraquartile range of 0.551 to 1.022 μg/mL compared to median concentration of 0.567 μg/mL with intraquartile range of 0.366 to 0.869 μg/mL for control plasma. 
Figure 3
 
TSP1 concentrations in the plasma of control and POAG patients. TSP1 is significantly higher in the POAG group (P < 0.0001). Individual data points (blue symbols) and superimposed box plots are presented as in Figure 1.
Figure 3
 
TSP1 concentrations in the plasma of control and POAG patients. TSP1 is significantly higher in the POAG group (P < 0.0001). Individual data points (blue symbols) and superimposed box plots are presented as in Figure 1.
Plotting TGFβ1 versus TSP1 concentrations of each plasma sample revealed that concentrations of the two cytokines were significantly correlated (P < 0.0001) for both control and POAG groups (Figs. 4A, 4B). The slopes of the best-fit lines were not different between POAG and control (P = 0.33), suggesting that the extent of platelet degranulation was not different between groups. 
Figure 4
 
Correlation between TGFβ1 and TSP1. TGFβ1 versus TSP1 plots with best-fit lines for the control (A) and POAG (B) plasma samples reveal significant linear correlation (P < 0.0001). Equation of the best-fit line and R 2 value are shown in the upper right corner of each plot.
Figure 4
 
Correlation between TGFβ1 and TSP1. TGFβ1 versus TSP1 plots with best-fit lines for the control (A) and POAG (B) plasma samples reveal significant linear correlation (P < 0.0001). Equation of the best-fit line and R 2 value are shown in the upper right corner of each plot.
To investigate the extent to which TGFβ1 and TSP1 in the plasma samples originates from platelet degranulation, experiments were performed in which varying doses of the platelet activator peptide TRAP6 were added to whole blood samples from four control patients prior to plasma preparation. Stimulation with TRAP6 resulted in a dose-dependent increase in TGFβ1 and TSP1 (Figs. 5A, 5B). Plotting TGFβ1 versus TSP1 concentrations of the TRAP6-stimulated plasma samples (Fig. 5C) revealed significant correlation (P < 0.0001) but with a slope of the best-fit line significantly different from the slopes of the best-fit lines of the nonstimulated plasma data (P < 0.001, Figs. 4A, 4B), suggesting that TGFβ1 and TSP1 are not solely derived from platelet degranulation. 
Figure 5
 
Correlation between TGFβ1 and TSP1 in plasma from blood samples stimulated with platelet activator TRAP6. Dose-dependent increases in TGFβ1 (A) and TSP1 (B) were found in samples from control donors (n = 4, not included in Figs. 1 1552 15524) that were stimulated with 0, 0.1, 1, and 10 μM TRAP6. Each donor is represented by a different color symbol. Plotting TGFβ1 versus TSP1 for the TRAP6-stimulated samples (C) reveals linear correlation (P < 0.0001). Equation of the best-fit line and R 2 value are shown in the upper right corner. The best-fit line for control plasma samples shown in Figure 4A is indicated by a dashed line.
Figure 5
 
Correlation between TGFβ1 and TSP1 in plasma from blood samples stimulated with platelet activator TRAP6. Dose-dependent increases in TGFβ1 (A) and TSP1 (B) were found in samples from control donors (n = 4, not included in Figs. 1 1552 15524) that were stimulated with 0, 0.1, 1, and 10 μM TRAP6. Each donor is represented by a different color symbol. Plotting TGFβ1 versus TSP1 for the TRAP6-stimulated samples (C) reveals linear correlation (P < 0.0001). Equation of the best-fit line and R 2 value are shown in the upper right corner. The best-fit line for control plasma samples shown in Figure 4A is indicated by a dashed line.
Discussion
In this study, we discovered a significant elevation of systemic TGFβ1 in POAG patients as compared to controls (Fig. 1). This finding potentially places POAG within a group of diseases associated with elevated systemic TGFβ1 or elevated TGFβ signaling. A fairly recent body of literature has emerged connecting elevated TGFβ signaling or elevated systemic TGFβ1 with microfibril defects in a number of diseases. 3948 Neptune and coworkers originally identified this correlation in a known microfibril deficiency, Marfan syndrome, in which hyperactivated TGFβ signaling is found in affected tissues and systemic TGFβ1 is elevated. 39,40,63 Microfibril defects are a possible mechanism of the increased systemic TGFβ1 in POAG patients found in this investigation, and could also explain elevated TGFβ2 and TGFβ1 in the AH of POAG patients as shown in a number of studies. 1320  
Sequestration of latent TGFβs by microfibrils in the extracellular matrix is a central mechanism of regulation of TGFβ signaling. 3234 Microfibril deficiencies are associated with increased TGFβ signaling and higher systemic TGFβ1 concentrations, 3948 likely due to incomplete sequestration of TGFβ1. 39 Our previous identification of a mutation in a microfibril gene, ADAMTS10, as causative for POAG in dogs, 54,55 along with other studies showing microfibril genes associated with glaucoma, 5153,64 led us to form our microfibril hypothesis of glaucoma stating that microfibril defects could cause glaucoma. The microfibril hypothesis of glaucoma predicts elevated systemic TGFβ1 in glaucoma patients, as found in other diseases caused by microfibril deficiencies. 50,55 The finding of elevated systemic TGFβ1 in POAG patients in this study (Fig. 1) is consistent with this prediction, providing additional evidence supporting the microfibril hypothesis of glaucoma. 
Another potential source of plasma TGFβ1 is platelets, which are numerous and which contain relatively high amounts of TGFβ1 in their α-granules. 38 Platelet degranulation during blood draw can significantly contribute to TGFβ1 measured in plasma samples 9,65 and is practically impossible to completely eliminate during blood sampling in a clinical setting. In the present study we minimized in vitro platelet degranulation by avoiding trauma to the vein, removing tourniquets after blood collection was initiated, keeping blood samples on ice, and preparing plasma shortly after blood draw. To investigate possible contributions from platelets, we chose TSP1 as a marker of platelet degranulation because it is highly expressed in platelet granules 66 and has been used as a marker of platelet activation in other studies. 67,68 Thrombospondin-1 was significantly elevated in POAG plasma (Fig. 3), with a linear correlation between TGFβ1 and TSP1 in both POAG and control samples (Figs. 4A, 4B), suggestive of platelets contributing to plasma TGFβ1. 
To investigate the extent of possible contributions from platelets, whole blood from several control patients was stimulated with TRAP6 peptide, which is a potent platelet activator, 59 prior to plasma preparation; TRAP6 caused a dose-dependent increase in plasma TGFβ1 and TSP1 (Figs. 5A, 5B). A linear correlation was found between TGFβ1 and TSP1 in the plasma samples from TRAP6-treated blood, with approximately 75% of the variation of TGFβ1 accounted for by variation in TSP1 based on the square of the Pearson correlation coefficient (Fig. 5C). In patient plasma samples that were not stimulated (Figs. 4A, 4B), variation in TGFβ1 was less dependent on TSP1 levels (36% and 22% for controls and POAG, respectively) as compared to the TRAP6-stimulated samples (Fig. 5C). These results suggest that while platelet degranulation likely contributed to plasma TGFβ1 in the patient samples, additional nonplatelet sources contributed as well. Correlations between TGFβ1 and TSP1 were not significantly different between cases and controls (P = 0.33, Figs. 4A, 4B), suggesting that the elevated TGFβ1 in POAG plasma was not due to increased platelet degranulation. 
Our finding of elevated TSP1 in POAG (Fig. 3) may also be relevant to pathogenesis. In addition to being abundant in platelet granules, TSP1 is expressed by other cell types and has a rather complicated relationship with TGFβ1 and glaucoma. It is an activator of TGFβ1, 69 raising the possibility that levels of activated TGFβ1 may be increased in POAG, though we found activated TGFβ1 in plasma to be below detection in our assay. Expression of TSP1 has been shown to be induced by TGFβ1, 70 including in the trabecular meshwork, 71,72 suggesting a possible feedforward mechanism leading to elevated TSP1 and activated TGFβ1. In relation to glaucoma, TSP1 protein expression was reported to be higher in the trabecular meshwork of glaucoma patients, 71 and TSP1 mRNA expression is induced by stretch activation of cultured lamina cribrosa cells. 73 Furthermore, TSP1-null mice have lowered intraocular pressure, 74 suggesting a role for TSP1 in regulating intraocular pressure. The elevated TSP1 in POAG patients found in this study suggests that TSP1 could potentially contribute to POAG by itself or in concert with TGFβ1. 
In conclusion, we have found elevated levels of TGFβ1 in plasma from POAG patients, as predicted by our microfibril hypothesis of glaucoma. If microfibril defects cause POAG, therapeutic approaches developed for microfibril deficiencies with similar elevations of TGFβ1 may be applied to POAG. Further study is needed to establish the mechanisms of increased TGFβ1 in POAG and the relationship between POAG and microfibrils. 
Acknowledgments
We thank our patient volunteers for participating in this study. 
Supported by National Eye Institute Grants EY020894 (RWK) and EY022618 (MAB), Clinician-Scientist Award from the American Glaucoma Society (RWK), Departmental Unrestricted Award from Research to Prevent Blindness, Inc., and Vanderbilt Vision Research Center (P30EY008126). 
Disclosure: J. Kuchtey, None; J. Kunkel, None; L.G. Burgess, None; M.B. Parks, None; M.A. Brantley Jr, None; R.W. Kuchtey, None 
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Figure 1
 
Total TGFβ1 concentration in the plasma of control and POAG patients. TGFβ1 is significantly higher in the POAG group (P < 0.0001). Box plots are shown superimposed on individual data points (blue circles) with the lower and upper horizontal lines of the boxes representing the first and third quartiles, respectively, and the extent of the vertical lines above and below the boxes indicating the 90th and 10th percentiles, respectively.
Figure 1
 
Total TGFβ1 concentration in the plasma of control and POAG patients. TGFβ1 is significantly higher in the POAG group (P < 0.0001). Box plots are shown superimposed on individual data points (blue circles) with the lower and upper horizontal lines of the boxes representing the first and third quartiles, respectively, and the extent of the vertical lines above and below the boxes indicating the 90th and 10th percentiles, respectively.
Figure 2
 
Total TGFβ1 concentration in the plasma is not correlated with patient age for either controls ([A], P = 0.17) or POAG patients ([B], P = 0.79). Plasma total TGFβ1 concentration was plotted versus age at time of blood draw. Equation of the best-fit line and R 2 value are shown in the upper left corner of each plot.
Figure 2
 
Total TGFβ1 concentration in the plasma is not correlated with patient age for either controls ([A], P = 0.17) or POAG patients ([B], P = 0.79). Plasma total TGFβ1 concentration was plotted versus age at time of blood draw. Equation of the best-fit line and R 2 value are shown in the upper left corner of each plot.
Figure 3
 
TSP1 concentrations in the plasma of control and POAG patients. TSP1 is significantly higher in the POAG group (P < 0.0001). Individual data points (blue symbols) and superimposed box plots are presented as in Figure 1.
Figure 3
 
TSP1 concentrations in the plasma of control and POAG patients. TSP1 is significantly higher in the POAG group (P < 0.0001). Individual data points (blue symbols) and superimposed box plots are presented as in Figure 1.
Figure 4
 
Correlation between TGFβ1 and TSP1. TGFβ1 versus TSP1 plots with best-fit lines for the control (A) and POAG (B) plasma samples reveal significant linear correlation (P < 0.0001). Equation of the best-fit line and R 2 value are shown in the upper right corner of each plot.
Figure 4
 
Correlation between TGFβ1 and TSP1. TGFβ1 versus TSP1 plots with best-fit lines for the control (A) and POAG (B) plasma samples reveal significant linear correlation (P < 0.0001). Equation of the best-fit line and R 2 value are shown in the upper right corner of each plot.
Figure 5
 
Correlation between TGFβ1 and TSP1 in plasma from blood samples stimulated with platelet activator TRAP6. Dose-dependent increases in TGFβ1 (A) and TSP1 (B) were found in samples from control donors (n = 4, not included in Figs. 1 1552 15524) that were stimulated with 0, 0.1, 1, and 10 μM TRAP6. Each donor is represented by a different color symbol. Plotting TGFβ1 versus TSP1 for the TRAP6-stimulated samples (C) reveals linear correlation (P < 0.0001). Equation of the best-fit line and R 2 value are shown in the upper right corner. The best-fit line for control plasma samples shown in Figure 4A is indicated by a dashed line.
Figure 5
 
Correlation between TGFβ1 and TSP1 in plasma from blood samples stimulated with platelet activator TRAP6. Dose-dependent increases in TGFβ1 (A) and TSP1 (B) were found in samples from control donors (n = 4, not included in Figs. 1 1552 15524) that were stimulated with 0, 0.1, 1, and 10 μM TRAP6. Each donor is represented by a different color symbol. Plotting TGFβ1 versus TSP1 for the TRAP6-stimulated samples (C) reveals linear correlation (P < 0.0001). Equation of the best-fit line and R 2 value are shown in the upper right corner. The best-fit line for control plasma samples shown in Figure 4A is indicated by a dashed line.
Table
 
Patient Characteristics
Table
 
Patient Characteristics
Control POAG P Value*
Number of patients 150 148
Age, y, mean (SD) 66.3 (7.8) 66.2 (12.2) 0.94
Female, n (%) 82 (54.7) 89 (60.1) 0.35
Caucasian, n (%) 143 (95.3) 136 (91.9) 0.25
African American, n (%) 6 (4.0) 9 (6.1) 0.44
Asian, n (%) 1 (0.7) 3 (2.0) 0.37
Primary family history,† n (%) 12 (8.0) 41 (27.7) 8.00 × 10−6
Any family history,‡ n (%) 19 (12.7) 74 (50.0) 5.04 × 10−6
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