Raynaud's phenomenon (RP) is a poorly understood disorder characterized by recurrent episodes of protracted vasospasm on cold exposure (1). The disease may occur as a primary manifestation (primary RP) or as part of a constellation of findings in patients with diverse rheumatologic conditions (secondary RP) (2). Although abnormalities in endothelial function in conduit and resistance vessels and elevations in circulating vasoconstrictor mediators such as endothelin 1 (ET-1) have been described (3–5), these findings have not been consistent (6, 7). In addition, the precise etiologic role of these abnormalities in the pathogenesis of primary RP versus secondary forms of this disorder is not clear. Circulating ET-1 levels do not seem to correlate with the intensity of vasospasm and are not consistently elevated in RP (8). These differences may stem in part from studies comparing patient populations with different levels of disease severity, presence of underlying connective tissue disease, and concomitant vascular risk factors.
The loss of nitric oxide (NO), the hallmark of endothelial dysfunction, is an attractive possibility as an overall mechanism for the pathogenesis of RP, since perturbations in the NO pathway may have pleiotropic effects on multiple effector pathways that may be relevant in RP, including ET-1 (9). In this regard, although abnormalities of both conduit and microvascular endothelial function have been demonstrated in RP, the contribution of these 2 levels of circulation in explaining the differential features in primary and secondary RP requires further definition (3, 4, 10, 11). Recently, a novel circulating endogenous inhibitor of endothelial nitric oxide synthase (eNOS), called asymmetric dimethylarginine (ADMA), has been described (12). ADMA is synthesized in human endothelial cells and is believed to alter eNOS function in many ways, including inhibition of function, increases in endothelial oxidative stress, and alterations in substrate binding (13).
We hypothesized that individuals with RP secondary to connective tissue diseases, when compared with subjects with primary RP, have heightened sensitivity of upper extremity conduit and resistance vessels, and that these changes are correlated with increases in ET-1 and ADMA. The present study was undertaken to test this hypothesis.
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- PATIENTS AND METHODS
RP as a manifestation of connective tissue disease is frequent and often precedes other symptoms and signs (30, 31). The manifestations, severity, and prognosis of secondary RP are very different from those of primary RP and include progression to cutaneous ulcerations and tissue necrosis (30, 32). Accurate differentiation of secondary RP from primary RP is partly dependent on identification of pathophysiologic mechanisms and vascular abnormalities that are unique to these two disease states. To achieve this, comparisons of primary and secondary RP at relatively similar degrees of severity is important, because variations of disease severity and concomitant vascular risk factors may complicate the interpretation of observations (33).
Accordingly, the main objective of this study was to compare and contrast microvascular and macrovascular abnormalities in a cohort of patients with primary and secondary RP who were carefully matched for demographic, LDL, hemodynamic, and severity profiles. The main findings of this study were the following: no differences in endothelium-dependent or -independent function in conduit vessels between patients with primary and secondary RP; abnormal vasoconstrictor response of brachial arteries to cold in both patients with primary and those with secondary RP, with no differences in the intensity of vasoconstriction between patients with primary and secondary RP; abnormal microvascular function in patients with secondary RP, but not patients with primary RP, as evidenced by a blunted cutaneous response to a reactive hyperemic stimulus; and increases in circulating ET-1 and ADMA in patients with secondary RP compared with patients with primary RP, accompanied by increases in VCAM-1 and MCP-1 levels.
To our knowledge, this is the first study to compare and contrast vasodilator mechanisms at both the resistance and conduit levels in primary and secondary RP. Our patient population with secondary RP was a heterogeneous group composed of patients with different connective tissue diseases that can present with RP symptoms. These individuals were compared with a cohort of patients with primary RP who were otherwise matched for disease severity and risk factors known to modulate vascular function. Indeed, blood pressure, LDL cholesterol, and adiposity were identical in the 2 groups. Conduit endothelial function and endothelium-independent function in our study, as determined by reactive hyperemia and responsiveness to cold in the brachial artery, did not differ between the 2 cohorts. However, both primary and secondary RP were characterized by abnormal vasoconstrictor responses to cold as opposed to the normal vasodilator response, reiterating abnormal sympathetic outflow at the vessel wall in this disease (34).
In contrast to the homogeneity of responses of conduit vessels in patients with primary and secondary RP, there was an impairment of microvascular responses in patients with secondary RP compared with patients with primary RP, as evidenced by a diminution in reactive hyperemic flow over time, adjusted for baseline perfusion. In our experience, AUC measurements, which represent a composite measurement over time, were superior to single-point measurements such as peak perfusion ratio and percent reactive hyperemia or time to peak. Consistent with prior studies, we demonstrated an increase in circulating ET-1 levels in patients with secondary RP compared with patients with primary RP (5, 35).
A novel finding in this study was the demonstration of elevation of a circulating endogenous inhibitor of NOS, ADMA, in patients with secondary RP compared with patients with primary RP. ADMA has been previously reported to be elevated in a variety of conditions associated with attenuation of arterial vasodilator reserve, including atherosclerosis and hypercholesterolemia (12, 36). In these conditions, ADMA levels are only elevated 0.5–3-fold above the normal concentrations. Thus, the range of values noted in these conditions is fairly small. Previous studies have shown that exogenous concentrations between 1 and 10 μM/liter affect endothelial-dependent vasodilation in rat mesenteric and cerebral vessels (37, 38). Similarly, incubation of cultured endothelial cells with oxidized LDL results in increases of ADMA from 0.6 μM/liter to 1.1 μM/liter at the end of a 48-hour period (39). Thus, ADMA levels are tightly regulated and the levels reported are likely to be of pathophysiologic significance. Both ADMA and ET-1, either alone or potentially together, can modulate resistance vessel function (40, 41).
In this regard, previous studies in experimental heart failure have shown that ET-1 stimulates ADMA (42), and our studies confirm a connection between the ET-1 axis and ADMA in the RP population. Inhibition of NOS in the skin with inhibitors such as L-NG-nitroarginine methyl ester can potentiate the effect of ET-1–mediated vasoconstriction, and thus these two mediators can potentially act together (43). Increases in ET-1 are well known to interact negatively with the NOS pathway and, together with elevations in ADMA, could represent a potent vasoconstrictor combination that sets the stage for progression of vascular disease and its complications. Indeed, ET-1 and ADMA levels taken together significantly correlated negatively with microvascular perfusion. Thus, it may be argued that the vascular abnormalities observed in patients with RP are unlikely to be affected by targeting one pathway alone.
Since ADMA has been reported to be increased in patients with renal failure and hypercholesterolemia, it is important to mention that none of the patients studied had renal dysfunction (creatinine >1.4 mg/dl) or elevations in LDL cholesterol (44). As expected, an inactive structural isomer of ADMA, SDMA, was not elevated in patients with secondary RP, supporting the accuracy of our ADMA analysis by high-performance liquid chromatography. Although increases in ET-1 and ADMA were negatively correlated with changes in microvascular flow, they did not correlate with conduit endothelial function (FMD%) or responses to cold in patients with either primary or secondary RP. This finding may relate to the fact that once other traditional risk factors that more directly correlate with conduit endothelial function (such as LDL cholesterol) are accounted for, conduit responses in RP are comparable, despite differential elevations of ADMA and ET-1. Thus, differences in microvascular flow may represent an inherent susceptibility of the cutaneous resistance circulation to these mediators in patients with secondary RP.
Previous studies have shown that ET-1 release and expression are linked to alterations in chemokine and adhesion molecule expression (45, 46). Our findings reaffirm and extend these observations linking the inflammatory cytokine network with the neurohormonal system, which may promote peripheral inflammation and oxidative stress in the cardiovascular system of patients with connective tissue disease and RP. The elevation of MCP-1 and VCAM-1 in patients with secondary RP may reflect the unfavorable effects of dysfunctional NOS and ET-1 pathways in patients with RP and indicate early activation of a proinflammatory process in the vasculature of patients with secondary RP. These findings add to evidence from prior studies demonstrating that patients with different connective tissue diseases associated with secondary RP have increased VCAM-1, ICAM-1, and MCP-1 levels, and that their levels in plasma correlate with their in situ expression (47).
In conclusion, both primary and secondary RP are characterized by abnormal conduit vessel vasoconstriction to cold. Secondary RP is also associated with attenuation of microvascular responses to reactive hyperemia and elevations in ADMA and ET-1. Increases in these vasoactive mediators acting together in patients with secondary RP may influence alterations in vascular function and proinflammatory pathways, and therefore, disease progression.