Marcel de Jong

Proliferation and extracellular matrix synthesis of smooth muscle cells cultured from human coronary atherosclerotic and restenotic lesions

OBJECTIVES The purpose of this study was to examine the proliferative capacity and extracellular matrix synthesis of human coronary plaque cells in vitro. BACKGROUND Common to both primary atherosclerosis and restenosis are vascular smooth muscle cell proliferation and production of extracellular matrix proteins. The applicability to humans of experimental animal models of these processes has been questioned. METHODS Primary atherosclerotic and restenotic lesions were excised by percutaneous directional coronary atherectomy in 93 patients. Smooth muscle cells were cultivated by an explant technique and identified by their morphology in culture, ultrastructural features under electron microscopy and immunostaining using monoclonal antibodies to smooth muscle cell alpha-actin. Proliferation in secondary culture was assessed with growth curves and the synthesis of collagen and sulfated glycosaminoglycans by the incorporation of 3H-proline and 35S-sulfate, respectively. These studies were also performed in cells derived from human umbilical artery media. RESULTS Success rates for primary (45%) and secondary (12%) culture of coronary cells were not influenced by clinical variables or lesion category. Primary culture success was improved by the presence of organized thrombus in the plaque and in relation to increased maximal cell density of the atherectomy specimen. Restenotic cells displayed more rapid growth than did cells of primary atherosclerotic origin, which grew in a manner similar to that of umbilical artery cells. Mean calculated population-doubling times for the three cell groups were 52 h (95% confidence interval [CI] 48 to 58 h), 71 h (95% CI 62 to 83 h) and 74 h (95% CI 65 to 84 h), respectively. Restenotic and primary atherosclerotic cells did not differ in the synthesis of collagen ([mean +/- SEM] 0.034 +/- 0.004 vs. 0.033 +/- 0.004 nmol isotope.microgram protein-1, p = NS) or sulfated glycosaminoglycans (11.47 +/- 1.07 vs. 15.37 +/- 3.10 nmol isotope.microgram protein-1, p = NS), but the coronary cells synthesized significantly more collagen and sulfated glycosaminoglycans than did umbilical artery cells (0.019 +/- 0.004 and 5.43 +/- 1.00 nmol isotope.microgram protein-1, respectively, both p < 0.05). CONCLUSIONS These data indicate that increased smooth muscle cell proliferation contributes to coronary restenosis in humans and support the concept that the extracellular matrix synthesis of adult smooth muscle cells is important to lesion formation.

Histologic characteristics of tissue excised during directional coronary atherectomy in stable and unstable angina pectoris

Unstable angina is an acute coronary syndrome associated with substantial short- and medium-term morbidity and mortality.1 The understanding of the pathogenesis of this syndrome has been based largely on postmortem studies of coronary arteries2 and supported by indirect evidence of coronary thrombosis in relation to the syndrome.3–5 Because directional coronary atherectomy is unique in extracting intact atheromatous tissue during coronary recanalization, it may facilitate the study of the processes taking place in the vessel in different coronary syndromes. In the present study the histopathologic characteristics of atherectomy samples retrieved in 93 patients with stable or unstable angina pectoris were compared and related to different clinical variables.

Triamcinolone acetonide activates an anti-inflammatory and folate receptor–positive macrophage that prevents osteophytosis in vivo

IntroductionTriamcinolone acetonide (TA) is used for osteoarthritis management to reduce pain, and pre-clinical studies have shown that TA limits osteophyte formation. Osteophyte formation is known to be facilitated by synovial macrophage activation. TA injections might influence macrophage activation and subsequently reduce osteophytosis. Although widely applied in clinical care, the mechanism through which TA exerts this effect remains unknown. In this animal study, we investigated the in vivo effects of TA injections on macrophage activation, osteophyte development and joint degeneration. Furthermore, in vitro macrophage differentiation experiments were conducted to further explain working mechanisms of TA effects found in vivo.MethodsOsteoarthritis was induced in rat knees using papain injections and a running protocol. Untreated and TA-treated animals were longitudinally monitored for 12 weeks with in vivo micro–computed tomography (μCT) to measure subchondral bone changes. Synovial macrophage activation was measured in vivo using folate receptor β (FRβ)-targeted single-photon emission computed tomography/computed tomography. Articular cartilage was analyzed at 6 and 12 weeks with ex vivo contrast-enhanced μCT and histology. To further explain the outcomes of our in vivo study, TA on macrophages was also studied in vitro. These cultured macrophages were either M1- or M2-activated, and they were analyzed using fluorescence-activated cell sorting for CD163 and FRβ expression as well as for messenger RNA (mRNA) expression of interleukin (IL)-10.ResultsOur in vivo study showed that intra-articular injections with TA strongly enhanced FRβ+ macrophage activation. Despite stimulated macrophage activation, osteophyte formation was fully prevented. There was no beneficial effect of TA against cartilage degradation or subchondral bone sclerosis. In vitro macrophage cultures showed that TA strongly induced monocyte differentiation towards CD163+ and FRβ+ macrophages. Furthermore, TA-stimulated M2 macrophages showed enhanced IL-10 expression at the mRNA level.ConclusionsTA injections potently induce a CD163+- and FRβ+-activated macrophage with anti-inflammatory characteristics such as reduced IL-10 production in vitro and lack of osteophytosis in vivo.

Clinical and histological determinants of smooth-muscle cell outgrowth in cultured atherectomy specimens: importance of thrombus organization.

Background:Coronary atherectomy provides a unique opportunity to obtain plaque tissue from a wide variety of clinical syndromes. We investigated the relation between the clinical status and histopathological substrate of tissue retrieved during directional coronary atherectomy and the proliferative and migratory potential of smooth-muscle cells judged from successful outgrowth during cell culture. Methods:After directional coronary atherectomy, tissue samples were examined macroscopically, divided into two equal pieces, and separately subjected to cell culture and histopathological study. Cell culture was performed using an explant technique. In-vitro smooth-muscle cell outgrowth was related to clinical and histological variables. Results:Atherosclerotic tissue was obtained from 98 consecutive atherectomy procedures. Histological examination revealed a broad spectrum of appearances, ranging from complex atheroma containing dense fibrous tissue, calcium deposits, macrophages, and necrotic debris to neointimal proliferation and organized thrombi. Smooth-muscle cell outgrowth was observed in 43 of the 98 samples (44%). Although not affected by any of the clinical variables, cell outgrowth was influenced by histological variables, in particular the presence of organizing thrombi. Outgrowth was successful in eight out of 10 samples with thrombus (80%) and in only 35 out of 88 (40%) without (P=0.03). Conclusion:The presence of organizing thrombi in the retrieved tissue facilitates smooth-muscle cell outgrowth and suggests an enhanced proliferative and migratory potential. These findings may be relevant to the understanding of neointimal proliferation in coronary syndromes where mural thrombosis is likely to occur.

Increased extracellular matrix synthesis by smooth-muscle cells obtained from in vivo restenotic lesions by directional coronary atherectomy

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Tumor uptake of 68Ga-DOTA-Tyr3-octreotate: animal PET studies of tumor flow and acute somatostatin receptor modulation in the CA20948 rat model

INTRODUCTION Factors determining the in vivo uptake of radiolabeled somatostatin analogs by neuroendocrine tumors are poorly known. The aim is to evaluate in vivo tumor perfusion and regulation of somatostatin receptors (sstr) following acute exposure to octreotide, in an animal model of neuroendocrine tumor. METHODS H(2)(15)O flow studies were performed in 8 CA20948 tumor-bearing rats and another 36 rats underwent three [(68)Ga]-DOTA-Tyr(3)-octreotate imaging sessions at 24-h intervals. After baseline (Day 0) imaging, scanning was repeated on Day 1 after octreotide injection (175 microg/kg), with a variable delay: no injection (controls, n=7), coinjection (n=6), and octreotide injection 20 min (n=7), 2 h (n=8) and 4 h (n=8) before imaging. Repeat images without octreotide was performed at Day 2 followed by sacrifice and tumor counting. RESULTS H(2)(15)O studies failed to measure quantitative tumor perfusion in this model. On Day 1, ratio of tumor uptake to Day 0 was 1.2+/-0.3 in controls; 0.6+/-0.2 in the coinjection group; 0.9+/-0.2, 1.1+/-0.1 and 1.2+/-0.2 in the other groups, respectively. Uptake in the coinjection group showed a statistically significant reduction of tumor uptake (P<.0001). All groups showed increased uptake on Day 2, without statistical differences between groups. In vivo tumor counts showed good correlation with ex vivo countings (R(2)=0.946). CONCLUSION Acute exposure to unlabeled octreotide in this neuroendocrine tumor model results in a rapid recycling or resynthesis of sstr. Positron emission tomography (PET) allowed to reliably assess quantitative uptake of [(68)Ga]-DOTA-Tyr(3)-octreotate over time in the same animal, but failed in this model to measure tumor perfusion.

Directional atherectomy: Combining basic research and intervention

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