Role of T Lymphocytes in Hypertension-Induced Cardiac Extracellular Matrix Remodeling

Methods

Results

The immune background was compared between C57 WT and BALB/c mice in Figure 1. Figure 1 shows that C57 WT mice are dominant TH1, and BALB/c mice are TH2 dominant. Moreover, the ratio of IFN-γ/IL-4 is 173 in the C57BL WT and 21 in the BALB/c, thus confirming their innate immune polarization. This murine strain difference in immune background has been characterized previously.¹⁰

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Figure 1. ELISA cytokine assay of C57 WT mice and BALB/c mice. Cultured splenocytes from C57 WT and BALB/c were assayed for IFN-γ, IL-4, and IL-10 to demonstrate the immunologic differences between the strains (n=3). *P<0.02.

The effect of chronic l-NAME administration on the left ventricular mechanics was compared among C57 SCID, C57 WT, and BALB/c mice (Table 2). Consistent with the report by Rossi et al,¹¹ chronic l-NAME administration did not induce cardiac hypertrophy. The maximum systolic pressure increased 31% in the C57 SCID mice (P<0.05), 34% (P<0.05) in the C57 WT mice, and 31% (P<0.05) in the BALB/c mice with l-NAME administration compared with their respective controls, which corresponds with the nonanesthetized systolic blood pressures shown in Figure 2. Correspondingly, arteriolar elastance significantly increased in all of the l-NAME–treated groups (P<0.05); however, there were no significant differences among all of the groups related to CO and SV compared with their respective controls. There was a mouse strain separation with the ventricular mechanical parameters. The end-diastolic volumes (Ved) and end-systolic volumes (Ves) were significantly increased in l-NAME–treated C57 SCID mice, decreased in BALB/c mice, and the Ves decreased in the C57 WT mice (P<0.05). The ejection fraction decreased 34% in the C57 SCID mice and increased 17% and 27% in the C57 WT and BALB/c mice, respectively (P<0.05). The response to the increased afterload is supported by the increased maximum rate of pressure increase over time, dP/dt_max and maximum rate of pressure decrease over time, dP/dt_min, in all of the groups (P<0.05), and stroke work (SW) and preload recruitable SW (the slope of SW plotted against V_ed) (PRSW) increased only in the C57 WT and BALB/c mice (P<0.05). The parameters for isovolumic contraction and relaxation, dP/dt_max-Ved and Tau Weiss, respectively, did not change with l-NAME administration. Most striking was the passive diastolic function parameter, β, which with the control C57 SCID and BALB/c mice was markedly less compared with the C57 WT (P<0.05). Additionally l-NAME administration decreased β in C57 SCID mice, from 0.15 to 0.08 mm Hg/μL (P<0.05), did not change in the C57 WT mice, and significantly increased, from 0.12 to 0.17 mm Hg/μL (P<0.05), in the BALB/c mice, when compared with their respective controls. In summary, those hemodynamic parameters related to the cardiac ECM were most affected by the mouse strain differences with l-NAME administration, because there were no differences in isovolumic contraction (dP/dt_max-Ved) and isovolumic relaxation (τ).

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Figure 2. The effect of l-NAME on systolic blood pressure measured with tail cuff in unanesthetized mice. A comparison in systolic blood pressure was made between control and those mice that had received l-NAME treatment. These data represent measurements made on days 27 to 29 (n=4). *P<0.0001 vs respective controls; †P<0.001 vs C57 WT l-NAME. ‡P<0.003 vs C57 WT control.

The comparison of the gene expression in cardiac tissues among the 6 groups with real-time PCR is shown in Table 3. Data were reported as the normalized threshold cycle number difference with the corresponding β-actin, and a lower threshold cycle number implies an increased gene expression. Comparison of the 3 control groups revealed that pro-collagen type I gene expression was higher in the C57 SCID and BALB/c compared with C57 WT, whereas the opposite was found with pro-MMP-9. Moreover the LOXL3 expression was increased in the control C57 SCID and decreased in the control BALB/c compared with the control C57 WT. l-NAME administration did not change procollagen type I and type III gene expressions compared with their respective controls. The pro-MMP-13 activity was increased in both C57 SCID and C57 WT mice (P<0.05), whereas pro-MMP-2 significantly increased in BALB/c mice with l-NAME administration (P<0.01). Moreover, l-NAME administration significantly decreased LOXL3 gene expression in C57 SCID mice (P<0.05), whereas LOXL3 increased in both C57 WT mice (P<0.05) and BALB/c mice (P<0.01). LOX gene expression also significantly increased (P<0.05) in BALB/c l-NAME–treated mice when compared with the BALB/c controls.

The Figure 3 shows that l-NAME administration increased pro-MMP-2 (P=0.005) and MMP-2 (P=0.05) activity in C57 SCID mice while decreasing these activities in C57 WT mice (P=0.012 and 0.01, respectively). In BALB/c mice, MMP-2 and MMP-9 were increased (P=0.04) when compared with the BALB/c control.

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Figure 3. Cardiac tissue MMP-9, pro-MMP-2, and MMP-2 activity with zymography. The cardiac MMP activities were determined after 30 days of l-NAME administration. The data represent the MMP activities related to the immune background with and without l-NAME treatment (n=4). *P<0.05 vs respective controls for MMP-9; †P<0.05 vs respective controls for pro-MMP-2, and ‡P<0.05 vs respective controls for MMP-2.

The fibrosing response to chronic hypertension was assessed by cardiac collagen content and collagen cross-linking. Table 4 demonstrates the ventricular collagen content measured by hydroxyproline. There were differences in collagen content when comparing the 3 control groups: the C57 SCID increased and the BALB/c decreased compared with the C57 WT control (P<0.05). The fibrillar collagen cross-linking in the BALB/c control was 21% less than that compared with the C57 WT and C57 SCID controls (P<0.05). l-NAME administration decreased total collagen content in C57 SCID mice (P=0.004), caused no change in C57 WT mice (P value not significant), and increased in the BALB/c (P=0.03). l-NAME administration decreased the cross-linked collagen in C57 SCID mice (P=0.006), caused no change in C57 WT (P value not significant), and increased in BALB/c mice (P=0.03). The percentage of cross-linked collagen in SCID mice was decreased from 57% to 32% (P=0.03). The alteration with cross-linking in the 3 groups explains the changes in Ves, Ved, and β after chronic l-NAME administration.

Figure 4 compares the LOX enzymatic activity in the study groups. l-NAME administration decreased LOX activity in C57 SCID mice (P=0.03) while increasing in BALB/c mice (P=0.02) and not changing in C57 WT mice (P value was not significant). Moreover, the correlative relationship between collagen cross-linking and ventricular stiffness (β) demonstrated R² at 0.5985 (P<0.01), as shown in Figure 5. This relationship reveals that maturated and stabilized collagen is a determinant of the diastolic function.

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Figure 4. Comparison of cardiac tissue LOX activity with l-NAME administration. The LOX activity was represented by the production of H₂O₂ and detected by Amplex Red oxidation from tissues harvested after 30 days of l-NAME treatment (n=4). *P<0.05 vs respective controls; †=P<0.05 vs C57 control.

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Figure 5. The correlation between left ventricular percentage of cross-linked collagen and ventricular stiffness (β).

Discussion

The present study hypothesized that mice with dissimilar T-helper immune backgrounds will remodel differently in response to chronic hypertension. Accordingly, the goal of this study was to examine the effects of chronic hypertension induced by l-NAME in mice that differ in T-lymphocyte function, namely C57 SCID, C57 WT, and BALB/c. The C57 SCID mice are devoid of functional T lymphocytes, and we demonstrated that the C57 WT are a predominant TH1 and the BALB/c mice are TH2, which is consistent with other reports.¹⁰ The important findings of this study are as follows. The cardiac output did not differ compared with the respective controls; however, there was a major separation among the 3 strains of mice related to cardiac ECM remodeling and ventricular mechanics affected by the ECM structure. The amount of ventricular collagen and the extent of cross-linking markedly differed when comparing the 3 groups of mice. Considerable differences in fibrillar cardiac collagen composition were noted among the different immune control groups. l-NAME administration did not alter cardiac ECM in the C57 WT; however, differences were evident the C57 SCID and BALB/c compared with the controls. Generally, the heart dilated in the C57 SCID phenotype in response to increased afterload was because of a diminution of collagen and cross-linking. C57 WT (TH1) demonstrated minimal structural changes, and, conversely, the BALB/c phenotype (TH2) demonstrated a decrease in ventricular volumes and increased ventricular collagen and cross-linking with increased afterload.

This study shows that the immune background of the mouse may affect the remodeling processes that occur in response to increased chronic afterload. Because the C57 SCID mice are of a C57 WT background, they are most comparable with the C57 WT except for lacking functional T lymphocytes. It is implicit that C57 WT mice are genetically different from the BALB/c. This difference, however, is a classical immunologic model to define host phenotype resistance or susceptibility, which are dependent on T-lymphocyte cytokines.¹² Consistent with our findings, Shi et al¹³ found that T-helper subsets accounted for the extent of hepatic fibrosis when comparing C57BL/6 and BALB/c with their respective SCID phenotypes. They demonstrated that C57BL/6 developed significantly less hepatic fibrosis after CCL₄-induced liver injury compared with the BALB/c with a similar degree of cellular necrosis. Given the lack of functional T and B lymphocytes in the SCID mice, our data and that of Shi et al¹³ strongly support the role of the lymphocyte in modulation of fibrosis. This concept is firmly supported by our recent observation in a SCID mouse model that failed to remodel subsequent to myocardial infarction; however, placement of a fibroblast patch graft on the infarcted site induced restorative remodeling and normal hemodynamic LV function.¹⁴

NOS inhibition with chronic administration of l-NAME induces a dose-dependent increase in systolic blood pressure that leads to cardiac and vascular remodeling.^11,15 However, this model has been shown by us and others not to result in cardiac hypertrophy.¹¹ Various authors have reported that chronic NO synthesis inhibition results in cardiac fibrosis, which corresponds with our cardiac collagen determinations.^11,16 The chronic hypertension induced by l-NAME may induce myocardial ischemia¹⁷ or, alternatively, change the mitochondrial function directly because of inhibition of NO relating to fibrosis.¹¹ However, we have demonstrated that there is a marked difference in cardiac collagen in mice with varied immune backgrounds that appears not to be related to that suggested above. Therefore, this model provides a good opportunity to investigate the regulatory role of T lymphocytes in the wall stress.

We suggest that cardiac collagen content and collagen cross-linking are associated with T-lymphocyte function. The fact that the C57 SCID mice exhibited a markedly differed response to the induced wall stress compared with C57 WT emphasizes that lymphocyte-derived cytokines play a role in cardiac collagen homeostasis. The gene expression after 30 days of l-NAME administration for pro-collagen I and III did not differ compared with controls. The MMP-9 and -13 gene expression and activities did not parallel the cardiac collagen content, which implies that the MMP activities are temporally altered during the course of cardiac remodeling and also do not represent the entire portfolio of cardiac MMPs. Moreover, the enzyme that mediates collagen cross-linking, LOX, also appeared to be affected by the differences in immune background. There are 2 LOX enzymes described in the cardiac tissue, LOX and LOXL3,¹⁸ of which LOX is the only one that has been shown to have collagen as a substrate.⁹ Although LOXL3 has been shown recently to be highly expressed in cardiovascular tissues, its substrate has yet to be proven (K. Csiszar, personal communication, July 2005). However, our data demonstrated that the LOX enzymatic function (Figure 3) parallels LOXL3 and not LOX gene expression (Table 3) and fibrillar collagen cross-linking (Table 4) in the 3 control and treatment groups. The increased cardiac collagen and collagen cross-linking paralleled the ventricular stiffness as also described by Badenhorst et al¹⁹ and, in addition our immunologically dissimilar mice, suggests that the ventricular stiffness is related to T-lymphocyte function.

Other reports have demonstrated that T-lymphocyte function affects cardiac diastolic function. We have shown previously with a TH2 murine model of HIV that there is significant diastolic dysfunction.² It is known that immunosuppressed transplanted cardiac and liver patients develop diastolic dysfunction.²⁰ Rodent studies exhibited markedly altered ECM composition with cyclosporine at clinical doses without other confounding factors, such as hypertension or renal dysfunction.²¹ Conversely, cardiac hypertrophy and fibrosis associated with hypertension have been related with altered immune function.^22–24 Activated T cells of both TH1 and TH2 subtypes can regulate fibroblasts activities by secreting IFN-γ and IL-4, respectively.^2,25 Most importantly, the role of TH1 cytokines in fibrotic conditions has been suggested to occur during the initiation of fibrosis and the TH2 cytokines during later stages.^4,26 We propose, therefore, that the TH1 immune responses may parallel the development of early compensatory fibrotic responses, whereas TH2 may constitute a latter phase of fibrotic remodeling in the heart.

Alternate T-lymphocyte subsets that may support our findings include regulatory T cells nTreg, Tr1, and TH3. Regulatory T cells (nTreg) CD4⁺CD25⁺ are functionally mature from the thymus, Tr1 are induced by IFN-γ from naïve T-cells and secrete IL-10, and TH3 lymphocytes are dominant sources of transforming growth factor-β1. The immunosuppressive activity of TH3 depends on the production of transforming growth factor-β1, which suppresses both TH1 and TH2 lymphocyte function.²⁷ Mast cells also regulate activities of fibroblasts through IL-4 secretion,²⁸ as do basophils²⁹ and eosinophils.³⁰ These cells, however, are under the direct control of TH1 and TH2 cytokines. The macrophages have also been described as mediators of cardiac fibrosis.³¹ In our model, however, SCID mice have functional macrophages,¹⁰ and, therefore, the C57BL/6 and C57 SCIDs should have responded in a similar fashion if macrophages were the primary mediators of fibrosis.