Leptin locally synthesized in carotid atherosclerotic plaques could be associated with lesion instability and cerebral emboli

Jacob Schneiderman, Katrin Schaefer, Frank D Kolodgie, Naphtali Savion, Shlomo Kotev-Emeth, Rima Dardik, Amos J Simon, Moshe Halak, Clara Pariente, Isaac Engelberg, Stavros Konstantinides, Renu Virmani, Jacob Schneiderman, Katrin Schaefer, Frank D Kolodgie, Naphtali Savion, Shlomo Kotev-Emeth, Rima Dardik, Amos J Simon, Moshe Halak, Clara Pariente, Isaac Engelberg, Stavros Konstantinides, Renu Virmani

Abstract

Background: Unstable carotid plaques cause cerebral emboli. Leptin promotes atherosclerosis and vessel wall remodeling. We hypothesized that carotid atherosclerotic lesion instability is associated with local leptin synthesis.

Methods and results: Carotid endarterectomy plaques from symptomatic (n=40) and asymptomatic patients with progressive stenosis (n=38) were analyzed for local expression of leptin, tumor necrosis factor (TNF)-α, and plasminogen activator inhibitor type 1. All lesions exhibited advanced atherosclerosis inclusive of thick- and thin-cap fibroatheromas or lesion rupture. Symptomatic lesions exhibited more plaque ruptures and macrophage infiltration (P=0.001 and P=0.05, respectively). Symptomatic plaques showed preferential leptin, TNF-α, and plasminogen activator inhibitor type 1 transcript (P=0.03, P=0.04, and P=0.05, respectively). Leptin mRNA and antigen in macrophages and smooth muscle cells were confirmed by in situ hybridization and immunohistochemistry. Plasma leptin levels were not significantly different between groups (P=1.0), whereas TNF-α was significantly increased in symptomatic patients (P=0.006). Human aortic smooth muscle cell culture stimulated by TNF-α, lipopolysaccharide, or lipoteichoic acid revealed 6-, 6.7-, and 6-fold increased secreted leptin antigen, respectively, at 72 hours (P<0.05).

Conclusions: Neurologically symptomatic patients overexpress leptin mRNA and synthesize leptin protein in carotid plaque macrophages and smooth muscle cells. Local leptin induction, presumably by TNF-α, could exert paracrine or autocrine effects, thereby contributing to the pathogenesis of lesion instability.

Clinical trial registration: URL: www.Clinicaltrials.gov. Unique identifier: NCT00449306.

Keywords: atherosclerosis; leptin; macrophages; smooth muscle cells; tumor necrosis factor-α.

Figures

Figure 1.
Figure 1.
Expression of leptin and its receptor (ObR) in a carotid fibroatheroma complicated by angiogenesis and intraplaque hemorrhage. A, Movat's pentachrome: a fibroatheromatous plaque with a large necrotic core (NC), intraplaque hemorrhage (HEM) (arrow), and overlying thick fibrous cap (FC). B, CD68-positive macrophages (MAC) within and surrounding the necrotic core. C, α-Actin-positive SMCs in the fibrous cap and remnants of media (arrowheads). D and E, Leptin (LEP) and its receptor (ObR), respectively. SMCs indicates smooth muscle cells.
Figure 2.
Figure 2.
Colocalization of SMCs and macrophages with leptin and its receptor (ObR) and with tumor necrosis factor-α (TNF-α) and its receptor (TNF-αR1) in a human carotid fibroatheroma. A, Dual staining for SMCs (brown-black) and leptin (LEP, red). B, SMCs near the fibrous cap represented by the area within the black box in (A) are positive for leptin. C, SMCs weakly positive for leptin receptors (ObR). D, Dual staining for macrophages (brown-black) and leptin (LEP, red). E and F, Region represented by the area within the black box in (D); macrophages are positive for both leptin and ObR. G, immunostain for TNF-α. H to K, Images within the black boxes in (G) correspond to SMC and macrophage-rich areas (MAC), respectively. TNF-α and its receptor (TNF-αR1) are expressed in both regions. SMCs indicates smooth muscle cells.
Figure 3.
Figure 3.
In situ hybridization analysis for leptin mRNA expression in carotid atherosclerotic plaques. A, Human carotid atherosclerotic lesion. Leptin mRNA expression in cells surrounding cholesterol clefts, presumably macrophages. B, Spindle-shaped cells, presumably SMCs, exhibit positive staining for leptin mRNA. C and D, Human adipose tissue, using antisense as positive control (C), or sense probe as negative control (D). Scale bars = 50 μm.
Figure 4.
Figure 4.
Real-time quantitative PCR analysis of leptin, TNF-α, and PAI-1 mRNA in symptomatic and asymptomatic plaques; plasma levels of leptin and TNF-α. A, Real-time quantitative PCR results for leptin mRNA in both groups were analyzed, revealing a significant difference (P=0.03). B, Similar analysis for TNF-α (P=0.044). C, Real-time quantitative PCR for PAI-1 mRNA (P=0.05). D, Plasma leptin levels were not significantly different in symptomatic and asymptomatic patients (P=1.0). E, Scattered plot of plasma leptin level vs body mass index for multiple individual patients of the whole series. The regression curve demonstrates that correlation is statistically significant (P<0.0001). F, TNF-α plasma levels were significantly higher in samples from symptomatic vs asymptomatic patients (P=0.006). Panels A through D show mean value and standard error.
Figure 5.
Figure 5.
Leptin antigen level in conditioned media from cultured human aortic SMCs upon stimulation by TNF-α, LPS or LTA. A, Representative experiment showing leptin immunoreactivity analyzed in conditioned media of aortic (Ao) SMCs after treatment with 50 ng/mL TNF-α, 100 ng/mL LPS, or 10 μg/mL LTA, determined at 24, 48, and 72 hours. Values were compared to leptin antigen levels in conditioned media from control cultures. B, Quantitative PCR for leptin performed on RNA extracted from human aortic cell lysate of 72 hours samples. For all measurements, values were >5 standard errors higher than the control. Error bars represent standard errors of technical repeats. SMCs indicates smooth muscle cells.

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