Phan-Kiet Tran

Increased Intimal Hyperplasia and Smooth Muscle Cell Proliferation in Transgenic Mice With Heparan Sulfate–Deficient Perlecan

Abstract— Smooth muscle cell (SMC) proliferation is a critical process in vascular disease. Heparan sulfate (HS) proteoglycans inhibit SMC growth, but the role of endogenous counterparts in the vessel wall in control of SMC function is not known in detail. Perlecan is the major HS proteoglycans in SMC basement membranes and in vessel wall extracellular matrix (ECM). In this study, transgenic mice with HS-deficient perlecan were analyzed with respect to vascular phenotype and intimal lesion formation. Furthermore, SMC cultures were established and characterized with respect to morphology, immunocytochemical features, proteoglycan synthesis, proliferative capacity, and ECM binding of basic fibroblast growth factor (FGF-2). In vitro, mutant SMCs formed basement membranes with perlecan core protein, but with decreased levels of HS, they showed diminished secretion of HS-containing perlecan into the medium and a defective ECM-binding capacity of FGF-2. In vitro, mutant SMCs showed increased proliferation compared with wild-type cells, and in vivo, enhanced SMC proliferation and intimal hyperplasia were observed after flow cessation of the carotid artery in mutant mice. The results indicate that the endogenous HS side-chains of perlecan contribute to SMC growth control both in vitro and during intimal hyperplasia, possibly by sequestering heparin-binding mitogens such as FGF-2.

Heparan Sulfate in Perlecan Promotes Mouse Atherosclerosis: Roles in Lipid Permeability, Lipid Retention, and Smooth Muscle Cell Proliferation

Heparan sulfate (HS) has been proposed to be antiatherogenic through inhibition of lipoprotein retention, inflammation, and smooth muscle cell proliferation. Perlecan is the predominant HS proteoglycan in the artery wall. Here, we investigated the role of perlecan HS chains using apoE null (ApoE0) mice that were cross-bred with mice expressing HS-deficient perlecan (Hspg2&Dgr;3/&Dgr;3). Morphometry of cross-sections from aortic roots and en face preparations of whole aortas revealed a significant decrease in lesion formation in ApoE0/Hspg2&Dgr;3/&Dgr;3 mice at both 15 and 33 weeks. In vitro, binding of labeled mouse triglyceride-rich lipoproteins and human LDL to total extracellular matrix, as well as to purified proteoglycans, prepared from ApoE0/Hspg2&Dgr;3/&Dgr;3 smooth muscle cells was reduced. In vivo, at 20 minutes influx of human 125I-LDL or mouse triglyceride-rich lipoproteins into the aortic wall was increased in ApoE0/Hspg2&Dgr;3/&Dgr;3 mice compared to ApoE0 mice. However, at 72 hours accumulation of 125I-LDL was similar in ApoE0/Hspg2&Dgr;3/&Dgr;3 and ApoE0 mice. Immunohistochemistry of lesions from ApoE0/Hspg2&Dgr;3/&Dgr;3 mice showed decreased staining for apoB and increased smooth muscle &agr;-actin content, whereas accumulation of CD68-positive inflammatory cells was unchanged. We conclude that the perlecan HS chains are proatherogenic in mice, possibly through increased lipoprotein retention, altered vascular permeability, or other mechanisms. The ability of HS to inhibit smooth muscle cell growth may also influence development as well as instability of lesions.

Control of smooth muscle cell proliferation in vascular disease.

Purpose of review Smooth muscle cell proliferation has previously been regarded as a central feature in vascular disease. The role of this process has recently been substantially re-evaluated, and we have reconsidered the functional importance of smooth muscle cell proliferation, the origin of proliferating smooth muscle cells in lesions, and the mechanisms whereby smooth muscle cell proliferation is controlled. In this review, we summarize recent progress in the understanding of smooth muscle cell proliferation, with a particular focus on how interactions between the extracellular matrix, smooth muscle cells, and mitogens control critical steps in this process. Recent findings Irrespective of the origin of smooth muscle cells in vascular lesions, fundamental interactions between the extracellular matrix and cell surface integrins are necessary in order to initiate a proliferative response in a quiescent smooth muscle cell, in a similar manner to any non-malignant cell. These interactions trigger intracellular signaling and cell cycle entry, which facilitate cell cycle progression and proliferation by mitogens. In addition, extracellular matrix interactions may also control the availability and activity of growth factors such as heparin-binding mitogens, which can be sequestered by heparan sulfate containing extracellular matrix components and regulate smooth muscle cell proliferation. Summary New insights into mechanisms whereby the extracellular matrix takes part in the control of smooth muscle cell proliferation suggest a number of putative targets for future therapies that can be applied to increase plaque stability, prevent the clinical consequences of atherosclerosis and improve outcomes after interventional procedures and organ transplantation.

Antenatal imatinib treatment reduces pulmonary vascular remodeling in a rat model of congenital diaphragmatic hernia

The pathophysiology of congenital diaphragmatic hernia (CDH) is constituted by pulmonary hypoplasia and pulmonary hypertension (PH). We previously reported successful treatment with imatinib of a patient with CDH. This study examines the effect of antenatal imatinib administration on the pulmonary vasculature in a rat model of CDH. Pregnant rats were given nitrofen to induce CDH. Controls were given olive oil. Half of the CDH fetuses and half of the controls were treated with imatinib antenatally E17-E21, rendering four groups: Control, Control+Imatinib, CDH, and CDH+Imatinib. Lung sections were obtained for morphometry and immunohistochemistry, and protein was purified for Western blot. Effects of nitrofen and imatinib on Ki-67, caspase-3, PDGF-B, and PDGF receptors were analyzed. Imatinib significantly reduced medial wall thickness in pulmonary arteries of rats with CDH. It also normalized lumen area and reduced the proportion of fully muscularized arteries. Imatinib also caused medial thinning in the control group. Cell proliferation was increased in CDH, and this proliferation was significantly reduced by imatinib. PDGF-B and PDGFR-β were upregulated in CDH, and imatinib treatment resulted in a downregulation. PDGFR-α remained unchanged in CDH but was significantly downregulated by imatinib. Antenatal imatinib treatment reduces development of medial wall thickness and restores lumen area in pulmonary arteries in nitrofen-induced CDH. The mechanism is reduced cell proliferation. Imatinib is an interesting candidate for antenatal therapy for PH in CDH, but potential side effects need to be investigated and more specific targeting of PDGF signaling is needed.

Perlecan heparan sulfate deficiency impairs pulmonary vascular development and attenuates hypoxic pulmonary hypertension.

AIMSnExcessive vascular cell proliferation is an important component of pulmonary hypertension (PH). Perlecan is the major heparan sulfate (HS) proteoglycan in the vascular extracellular matrix. It binds growth factors, including FGF2, and either restricts or promotes cell proliferation. In this study, we have explored the effects of perlecan HS deficiency on pulmonary vascular development and in hypoxia-induced PH.nnnMETHODS AND RESULTSnIn normoxia, Hspg2(Δ3/Δ3) mice, deficient in perlecan HS, had reduced pericytes and muscularization of intra-acinar vessels. Pulmonary angiography revealed a peripheral perfusion defect. Despite these abnormalities, right ventricular systolic pressure (RVSP) and myocardial mass remained normal. After 4 weeks of hypoxia, increases in the proportion of muscularized vessels, RVSP, and right ventricular hypertrophy were significantly less in Hspg2(Δ3/Δ3) compared with wild type. The early phase of hypoxia induced a significantly lower increase in fibroblast growth factor receptor-1 (FGFR1) protein level and receptor phosphorylation, and reduced pulmonary artery smooth muscle cell (PASMC) proliferation in Hspg2(Δ3/Δ3). At 4 weeks, FGF2 mRNA and protein were also significantly reduced in Hspg2(Δ3/Δ3) lungs. Ligand and carbohydrate engagement assay showed that perlecan HS is required for HS-FGF2-FGFR1 ternary complex formation. In vitro, proliferation assays showed that PASMC proliferation is reduced by selective FGFR1 inhibition. PASMC adhesion to fibronectin was higher in Hspg2(Δ3/Δ3) compared with wild type.nnnCONCLUSIONSnPerlecan HS chains are important for normal vascular arborization and recruitment of pericytes to pulmonary vessels. Perlecan HS deficiency also attenuates hypoxia-induced PH, where the underlying mechanisms involve impaired FGF2/FGFR1 interaction, inhibition of PASMC growth, and altered cell-matrix interactions.

Pulmonary vein stenosis: Challenges ahead.

In this issue of the Journal, Quinonez and colleagues 1 report their experience with and treatment of 49 patients with pulmonary vein stenosis. This article takes on the very difficult challenge to map out the ‘‘relentless’’ nature of multivessel pulmonary vein stenosis and the multidisciplinary commitments that are required for its treatment. We are encouraged by their acceptable short-term survival—67% at 1 year—that is the result of good surgery and high awareness upon postoperative follow-up, where the threshold to catheterization and intervention was low. Low weight at surgery, early surgical repair, and somewhat intriguing right ventricle pressure <75% of systemic are considered risk factors with poor prognosis. When multiple strategies employed to solve a single problem have persistently disappointing results and a heavy burden of repeated surgeries and interventions, we need to ask ourselves what is wrong. Pulmonary vein stenosis is at its fundament a process of pathologic cell proliferation and deposition of large amounts of extracellular matrix. 2 Despite the rare occurrence and difficulties in retrieving human tissue for basic research, enlightening knowledge has been generated. Riedlinger and colleagues 3 showed that platelet-derived growth factors A and B, vascular endothelial growth factor, fibroblast growth factor, and activated receptor tyrosine kinases are involved; and Kato and colleagues 4,5 demonstrated that transforming growth factor beta-1, a stimulator of matrix expansion, is upregulated in human pulmonary vein stenosis. Is this type of basic science important? Are itsfindings important? What does itmeanin terms of the management of recurrent pulmonary vein stenosis? Acceptingtheshortcomingsofsurgeryandcatheterinterventions, it was particularly uplifting to read the Discussion offered by Quinonez and colleagues, where it was revealed that 59% of the patients in their study were also being enrolled in another trial to test the effect of imatinib and bevacizumab. The choices of these drugs were not made at random. The former inhibits tyrosine kinases, and the latter is an antibody that inhibits vascular endothelial growth factor. A brief comment on preliminary data reveals that the treatments had no effect on survival. Obviously, we still have many challenges ahead. In the meantime, we can learn from Quinonez and colleagues’ purposeful and tenacious approach to a very difficult problem. These are the hallmarks of pediatric cardiac surgery. We shall wait with patience for the results of the coming studies.

Midterm results of the Ross procedure in children : An appraisal of the subannular implantation with interrupted sutures technique

OBJECTIVES The support of the pulmonary autograft root by the fibromuscular left ventricular outflow tract is emphasized to address the concern related to the dilatation of the pulmonary autograft structures in the paediatric population. METHODS This retrospective study analyses the outcomes of 75 children who were operated between 1998 and 2012 with the subannular interrupted sutures technique at a median age of 10.2 years (range, 5.3 months‐18.0 years). Median follow‐up time was 5.2 years (range, 3 days‐13.2 years). RESULTS There were no deaths, but there were 3 reinterventions on the autograft for regurgitation and 2 resections of left ventricular outflow tract obstruction. There was no significant autograft stenosis, and freedom from moderate‐to‐severe regurgitation was 95% (95% confidence interval: 89‐100) and 88% (95% confidence interval: 77‐99) at 5 and 10 years, respectively. Median z‐scores at the latest follow‐up examination were, at the annulus, 0.31 [interquartile range (IQR) = −0.81 to 1.2]; at the sinus of Valsalva, 2.7 (IQR = 1.5‐3.5); and at the sinotubular junction, 3.1 (IQR = 1.7‐4.2). The correlation between z‐scores and time after the operation was negative at the level of the annulus (r = −0.29, P = 0.034) but positive at the level of the sinus (r = +0.37, P = 0.005) and the sinotubular junction (r = +0.26, P = 0.068). The median rate of change in the z‐score at the annulus was low, 0.065 z‐score/year (IQR = −0.13 to 0.43). CONCLUSIONS The subannular interrupted sutures implantation technique is associated with acceptable risks and, in the midterm, delivers limited annular dilatation, autograft regurgitation and delayed need for autograft reintervention.

Effect of Glyceryl Trinitrate on Staphylococcus aureus Growth and Leukocyte Activation during Simulated Extracorporeal Circulation

BACKGROUNDnPreviously, nitric oxide has been shown to possess antimicrobial effects. In this study, we aim to test the effect of glyceryl trinitrate (GTN) on Staphylococcus aureus growth during simulated extracorporeal circulation (SECC) and also to examine the effect of S. aureus, alone and in combination with GTN, on activation markers of the innate immune system during SECC.nnnMETHODSnIn an in vitro system of SECC, we measured GTN-induced changes in markers of leukocyte activation in whole blood caused by S. aureus infestation, as well as the effect of GTN on S. aureus growth.nnnRESULTSnGTN had no effect on S. aureus growth after 240 minutes SECC. Staphylococcus aureus reduced the expression of granulocyte Fcγ-receptor CD32 but stimulated the expression of monocyte CD32. Staphylococcus aureus stimulated expression of some leukocyte adhesion key proteins, activation marker CD66b, lipopolysaccharide-receptor CD14, and C3b-receptor CD35. Staphylococcus aureus and GTN addition induced significant increases in monocyte CD63 (lysosomal granule protein) levels.nnnCONCLUSIONnGTN does not affect S. aureus growth during SECC and has no effect on SECC-induced leukocyte activation.

Enhanced Growth of Staphylococcus aureus after Nitric Oxide Supplementation during Simulated Extracorporeal Circulation

BACKGROUNDnSeveral factors contribute to postoperative bacterial infections in cardiac surgery. Long operation times and the use of extracorporeal circulation increase the risk of infection. Nitric oxide has been shown to possess a broad spectrum antimicrobial effect.nnnMETHODSnIn this study, we investigated the effect of nitric oxide on S. AUREUS growth in whole blood during simulated extracorporeal circulation.nnnRESULTSnS. AUREUS growth increased 6.2-fold after 180 min SECC in the presence of nitric oxide. Leukocyte counts remained unchanged without any differences between the groups. We observed a steady increase in markers of oxidative stress and activity of the innate immune system. Myeloperoxidase levels increased 8-fold, and C3a and terminal complement complex by 2-fold after 180 min.nnnCONCLUSIONnS. AUREUS growth is not due to the effect of nitric oxide on the innate immune system but from its effect on the bacteria itself. It has been shown that nitric oxide stimulates the expression of inducible lactate dehydrogenase, specific to S. AUREUS, which improves its resistance to oxidative stress, and may give S. AUREUS a survival advantage resulting in increased growth.