Biomimetic MRI contrast agent for imaging of inflammation in atherosclerotic plaque of ApoE-/- mice: a pilot study.

OBJECTIVE: Atherosclerosis involves an inflammatory process characterized by cellular and molecular responses. A slow-clearance blood-pool paramagnetic agent (CMD-A2-Gd-DOTA: P717) chemically modified to create a functionalized product (F-P717) for targeting inflammation in vessel walls was evaluated in vivo in mice. METHODS AND RESULTS: Carboxylate and sulfate groups were grafted onto the macromolecular paramagnetic Gd-DOTA-dextran backbone. Products were also fluorescently labeled with rhodamine isothiocyanate. Pre- and postcontrast MRI was performed on a 2-Tesla magnet in ApoE-/- and control C57BL/6 mice after P717 or F-P717 injection at a dose of 60 micromol Gd/kg. Axial T1-weighted images of the abdominal aorta were obtained using a 2D multislice spin-echo sequence. F-P717 significantly enhanced the magnetic resonance imaging (MRI) signal in the abdominal aortic wall of ApoE-/- mice (>50% signal-to-noise ratio increase between 10 and 30 minutes), but not of control mice. P717 produced only moderate (<20%) MRI signal enhancement within the same time frame. The MRI data were correlated to histopathology. Immunofluorescence in ApoE-/- mice colocalized F-P717 but not P717 with the inflammatory area revealed by P-selectin labeling. CONCLUSION: This study demonstrates the efficacy of F-P717 as a new molecular imaging agent for noninvasive in vivo MRI location of inflammatory vascular tree lesions in ApoE-/- mice.

Increased insulin-stimulated expression of arterial angiotensinogen and angiotensin type 1 receptor in patients with type 2 diabetes mellitus and atheroma.

OBJECTIVE: Because inhibition of the renin-angiotensin system (RAS) reduces the onset of type 2 diabetes (T2D) and prevents atherosclerosis, we investigated the expression of RAS in the arterial wall of T2D and nondiabetic (CTR) patients. METHODS AND RESULTS: mRNA and protein levels of angiotensinogen (AGT), angiotensin-converting enzyme (ACE) and AT1 receptor (AT1R) were determined in carotid atheroma plaque, nearby macroscopically intact tissue (MIT), and in vascular smooth muscle cells (VSMCs) before and after insulin stimulation from 21 T2D and 22 CTR patients. AGT and ACE mRNA and their protein levels were 2- to 3-fold higher in atheroma and in MIT of T2D patients. VSMCs from T2D patients had respectively 2.5- and 5-fold higher AGT and AT1R mRNA and protein contents. Insulin induced an increase in AGT and AT1R mRNA with similar ED50. These responses were blocked by PD98059, an inhibitor of MAP-kinase in the two groups whereas wortmannin, an inhibitor of PI3-kinase, partially prevented the response in CTR patients. Phosphorylated ERK1-2 was 4-fold higher in MIT from T2D than from CTR patients. CONCLUSIONS: The arterial RAS is upregulated in T2D patients, which can be partly explained by an hyperactivation of the ERK1-2 pathway by insulin.

Reduced atherosclerotic lesion size in P-selectin deficient apolipoprotein E-knockout mice fed a chow but not a fat diet.

P-selectin. We investigated the role of P-selectin on the development of vascular lesions in an ApoE(-/-) male mice. Double-knockout (ApoE(-/-), P-selectin(-/-); DKO) were compared to single-knockout (ApoE(-/-); SKO) mice. They were fed a chow or fat diet for 3, 6, 15, and 20 weeks, without any differences in cholesterol levels. DKO mice fed a chow diet exhibited a ratio of lesion area over media lower than SKO mice, for 3 (P < .03) , 6 (P < .001), and 15 (P < .02) weeks. DKO mice fed a fat diet showed a lower ratio only at 3 weeks. P-selectin deficiency in ApoE(-/-) mice has a protective effect in atherosclerotic lesions development. Reduction of lesion size depends on diet type and duration. A fat diet could neutralize the beneficial effects of P-selectin deficiency, inducing atherosclerotic lesions via probably other adhesion molecules.

Induction of tissue kallikrein in human carotid atheroma does not lead to kallikrein-kinins pathway activation.

OBJECTIVE: The impairment of the tissue kallikrein-kinin system (KKS) may result in atheroma development. To determine the involvement of KKS in pathophysiology of human atherosclerosis, we examined the expression of all components of this system as well as angiotensinogen (another tissue kallikrein (TK) substrate), at messenger ribonucleic acid (mRNA) and protein levels in the human carotid artery with and without atheroma. METHODS: mRNA levels were compared with semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) between atheroma plaque and intact tissue obtained during carotid endarterectomy in 15 patients. The cellular localization of the transcripts and proteins was analyzed with in situ hybridization and immunohistochemistry. TK activity was measured using chromogenic substrate. RESULTS: The kininogen mRNA was not detected in carotid wall. The TK mRNA was increased four-fold and TK activity 23-fold in atheroma plaque compared with intact tissue. No difference was observed for B1, B2 receptors, kallistatin, angiotensinogen and protein-kinase G type 1alpha (PK-G) mRNAs. The TK and angiotensinogen transcripts as well as kininogen and angiotensinogen proteins were present in both intimal and medial cells. The kininogen immunoreactivity was weaker in atheroma. CONCLUSIONS: All KKS components were synthesized in arterial wall except kininogen probably coming from plasma. The absence of PK-G mRNA down-regulation in atheroma suggests that the kallikrein induction does not lead to KKS activation.