Vyacheslav A. Korshunov

Flow-Induced Vascular Remodeling in the Mouse: A Model for Carotid Intima-Media Thickening

Objective—Vascular remodeling of the carotid artery with intima-media thickness (IMT) is an important predictive factor for human cardiovascular disease. We characterized a mouse model of vascular remodeling. Methods and Results—The left external and internal carotid branches were ligated so that left carotid blood flow was reduced to flow via the occipital artery. In response to partial ligation of the left carotid artery (LCA), blood flow significantly decreased (−90%) in the LCA and increased (+70%) in the right carotid artery (RCA). Morphometry showed that both RCA and LCA underwent outward remodeling that was maximal at one week. Remodeling was greater in the RCA with predominantly increased lumen and very little increase in media or adventitia. In the LCA there was a dramatic increase in media with adventitia growth and intima formation. Correlation analysis indicated that the outward remodeling was more likely due to primary changes in the vessel wall rather than to changes in the lumen, such as shear stress. Mechanistic studies suggested roles for macrophage infiltration, upregulation of matrix metalloproteinase (MMP)-9, extracellular matrix reorganization, and vascular smooth muscle cell proliferation in LCA remodeling. Conclusions—The mouse model described here may be useful to define genetic determinants of IMT and identify new targets for therapy based on vascular remodeling.

Vascular Remodeling Hemodynamic and Biochemical Mechanisms Underlying Glagov’s Phenomenon

An important concept for vascular remodeling, termed Glagov’s phenomenon, is that arteries remodel to maintain constant flow despite increases in atherosclerotic lesion mass. Although Glagov’s phenomenon was originally described only for the case of arterial remodeling in response to growth of atherosclerotic plaques, experimental and clinical observations indicate that blood flow properties influence remodeling after angioplasty, hypertension, and flow diversion as well as atherosclerotic plaque progression. This review attempts to define Glagov’s observation in terms of the physical parameters of blood in conduit arteries that must determine the remodeling response. Next we review experiments that have begun to identify specific molecules that influence vascular remodeling and therefore may serve as mediators for the phenomena. More comprehensive analyses of the specific molecular pathways in the vessels that determine constant flow may provide new therapeutic approaches to regulate vascular remodeling.

Fibronectin Is an Important Regulator of Flow-Induced Vascular Remodeling

OBJECTIVE Fibronectin is an important regulator of cell migration, differentiation, growth, and survival. Our data show that fibronectin also plays an important role in regulating extracellular matrix (ECM) remodeling. Fibronectin circulates in the plasma and is also deposited into the ECM by a cell dependent process. To determine whether fibronectin affects vascular remodeling in vivo, we asked whether the fibronectin polymerization inhibitor, pUR4, inhibits intima-media thickening, and prevents excess ECM deposition in arteries using a mouse model of vascular remodeling. METHODS AND RESULTS To induce vascular remodeling, partial ligation of the left external and internal carotid arteries was performed in mice. pUR4 and the control peptide were applied periadventitially in pluronic gel immediately after surgery. Animals were euthanized 7 or 14 days after surgery. Morphometric analysis demonstrated that the pUR4 fibronectin inhibitor reduced carotid intima (63%), media (27%), and adventitial thickening (40%) compared to the control peptide (III-11C). Treatment with pUR4 also resulted in a dramatic decrease in leukocyte infiltration into the vessel wall (80%), decreased ICAM-1 and VCAM-1 levels, inhibited cell proliferation (60% to 70%), and reduced fibronectin and collagen I accumulation in the vessel wall. In addition, the fibronectin inhibitor prevented SMC phenotypic modulation, as evidenced by the maintenance of smooth muscle (SM) alpha-actin and SM myosin heavy chain levels in medial cells. CONCLUSIONS These data are the first to demonstrate that fibronectin plays an important role in regulating the vascular remodeling response. Collectively, these data suggest a therapeutic benefit of periadventitial pUR4 in reducing pathological vascular remodeling.

Axl, A Receptor Tyrosine Kinase, Mediates Flow-Induced Vascular Remodeling

Intima-media thickening (IMT) in response to hemodynamic stress is a physiological process that requires coordinated signaling among endothelial, inflammatory, and vascular smooth muscle cells (VSMC). Axl, a receptor tyrosine kinase, whose ligand is Gas6, is highly induced in VSMC after carotid injury. Because Axl regulates cell migration, phagocytosis and apoptosis, we hypothesized that Axl would play a role in IMT. Vascular remodeling in mice deficient in Axl (Axl−/−) and wild-type littermates (Axl+/+) was induced by ligation of the left carotid artery (LCA) branches maintaining flow via the left occipital artery. Both genotypes had similar baseline hemodynamic parameters and carotid artery structure. Partial ligation altered blood flow equally in both genotypes: increased by 60% in the right carotid artery (RCA) and decreased by 80% in the LCA. There were no significant differences in RCA remodeling between genotypes. However, in the LCA Axl−/− developed significantly smaller intima+media compared with Axl+/+ (31±4 versus 42±6×10−6 &mgr;m3, respectively). Quantitative immunohistochemistry of Axl−/− LCA showed increased apoptosis compared with Axl+/+ (5-fold). As expected, p-Akt was decreased in Axl−/−, whereas there was no difference in Gas6 expression. Cell composition also changed significantly, with increases in CD45+ cells and decreases in VSMC, macrophages, and neutrophils in Axl−/− compared with Axl+/+. These data demonstrate an important role for Axl in flow-dependent remodeling by regulating vascular apoptosis and vascular inflammation.

Strain-Dependent Vascular Remodeling The “Glagov Phenomenon” Is Genetically Determined

Background—Atherosclerosis of the carotid artery, called intima-media thickening (IMT), is a form of vascular remodeling that is an important predictor for cardiovascular events and has a strong genetic component. Methods and Results—Recently, we established a mouse model of vascular remodeling based on partial ligation of the carotid, which is relevant to the “Glagov phenomenon.” We hypothesized that there would be genetically determined differences in outward remodeling and IMT induced by carotid flow alterations. We compared vascular remodeling among 5 inbred strains of mice. Despite similar changes in flow among the strains in the left carotid artery (LCA), we observed dramatic differences in remodeling of the partially ligated LCA relative to control. The smallest IMT volume (26±3 μm3) was found in C3H/HeJ mice, and the largest were in SJL/J (59±10 μm3) and FVB/NJ (81±6 μm3). Shear stress did not differ after ligation among strains. Lumen area decreased only when stenosis was ≥55%. IMT correlated significantly with outward remodeling among inbred strains (except C3H). There were significant strain-dependent differences in remodeling index (measured as vessel area/IMT), which suggest fundamental alterations in sensing or transducing hemodynamic signals among strains. Among hemodynamic factors, low shear stress and high heart rate were predictive for IMT. Specifically, heart rate (bpm: C3H, 592±6; SJL, 649±6; FVB, 683±7) but not systolic blood pressure (mm Hg: C3H, 116±2; SJL, 119±1; FVB, 136±1) was predictive. Conclusions—The present study indicates that performing a genetic cross of these strains and total genome scan should identify genes that mediate vascular remodeling.

Role of Nuclear Ca2+/Calmodulin-Stimulated Phosphodiesterase 1A in Vascular Smooth Muscle Cell Growth and Survival

In response to biological and mechanical injury, or in vitro culturing, vascular smooth muscle cells (VSMCs) undergo phenotypic modulation from a differentiated “contractile” phenotype to a dedifferentiated “synthetic” one. This results in the capacity to proliferate, migrate, and produce extracellular matrix proteins, thus contributing to neointimal formation. Cyclic nucleotide phosphodiesterases (PDEs), by hydrolyzing cAMP or cGMP, are critical in the homeostasis of cyclic nucleotides that regulate VSMC growth. Here, we demonstrate that PDE1A, a Ca2+-calmodulin–stimulated PDE preferentially hydrolyzing cGMP, is predominantly cytoplasmic in medial “contractile” VSMCs but is nuclear in neointimal “synthetic” VSMCs. Using primary VSMCs, we show that cytoplasmic and nuclear PDE1A were associated with a contractile marker (SM-calponin) and a growth marker (Ki-67), respectively. This suggests that cytoplasmic PDE1A is associated with the “contractile” phenotype, whereas nuclear PDE1A is with the “synthetic” phenotype. To determine the role of nuclear PDE1A, we examined the effects loss-of-PDE1A function on subcultured VSMC growth and survival using PDE1A RNA interference and pharmacological inhibition. Reducing PDE1A function significantly attenuated VSMC growth by decreasing proliferation via G1 arrest and inducing apoptosis. Inhibiting PDE1A also led to intracellular cGMP elevation, p27Kip1 upregulation, cyclin D1 downregulation, and p53 activation. We further demonstrated that in subcultured VSMCs redifferentiated by growth on collagen gels, cytoplasmic PDE1A regulates myosin light chain phosphorylation with little effect on apoptosis, whereas inhibiting nuclear PDE1A has the opposite effects. These suggest that nuclear PDE1A is important in VSMC growth and survival and may contribute to the neointima formation in atherosclerosis and restenosis.

Hydrogen Peroxide Activates the Gas6-Axl Pathway in Vascular Smooth Muscle Cells

Axl, a receptor tyrosine kinase, is involved in cell survival, proliferation, and migration. We have shown that Axl expression increases in the neointima of balloon-injured rat carotids. Because oxidative stress is known to play a major role in remodeling of injured vessels, we hypothesized that H2O2 might activate Axl by promoting autophosphorylation. H2O2 rapidly stimulated Axl tyrosine phosphorylation in rat vascular smooth muscle cells within 1 min that was maximal at 5 min (6-fold). The response to H2O2 was concentration-dependent with EC50 of ∼500 μm. Axl phosphorylation was partly dependent on production of its endogenous ligand, growth arrest gene 6 (Gas6), because Axl-Fc, a fragment of Axl extracellular domain that neutralizes Gas6, inhibited H2O2-induced Axl phosphorylation by 50%. Axl phosphorylation by H2O2 was also attenuated by warfarin, which inhibits Gas6 activity by preventing post-translational modification. In intact vessels Axl was phosphorylated by H2O2, and Axl phosphorylation was inhibited by warfarin treatment in balloon-injured carotids. Akt, a downstream target of Axl, was phosphorylated by H2O2in Axl+/+ mouse aorta but significantly inhibited in Axl-/- aorta. Intimal proliferation was decreased significantly in a cuff injury model in Axl-/- mice compared with Axl+/+ mice. In summary, Axl is an important signaling mediator for oxidative stress in cultured vascular smooth muscle cells and intact vessels and may represent an important therapeutic target for vascular remodeling and response to injury.

Axl-dependent signalling: a clinical update.

Axl is a receptor tyrosine kinase that was originally cloned from cancer cells. Axl belongs to the TAM (Tyro3, Axl and Mertk) family of receptor tyrosine kinases. Gas6 (growth-arrest-specific protein 6) is a ligand for Axl. Activation of Axl protects cells from apoptosis, and increases migration, aggregation and growth through multiple downstream pathways. Up-regulation of the Gas6/Axl pathway is more evident in pathological conditions compared with normal physiology. Recent advances in Axl receptor biology are summarized in the present review. The emphasis is given to translational aspects of Axl-dependent signalling under pathological conditions. In particular, inhibition of Axl reduces tumorigenesis and prevents metastasis as well. Axl-dependent signals are important for the progression of cardiovascular diseases. In contrast, deficiency of Axl in innate immune cells contributes to the pathogenesis of autoimmune disorders. Current challenges in Axl biology are related to the functional interactions of Axl with other members of the TAM family or other tyrosine kinases, mechanisms of ligand-independent activation, inactivation of the receptor and cell-cell interactions (with respect to immune cells) in chronic diseases.

Smooth muscle apoptosis and vascular remodeling.

Purpose of reviewThe present review is to summarize recent advances in molecular mechanisms that regulate vascular smooth muscle cell apoptosis during vascular remodeling. In normal blood vessels apoptosis counteracts cell division, whereas apoptosis is especially crucial for regulating vascular remodeling during cardiovascular diseases. Recent findingsRecent results have expanded our knowledge regarding the signaling pathways and molecules that regulate vascular smooth muscle cell death in postnatal vascular remodeling. Compelling data from genetic disorders associated with vascular smooth muscle cell loss (e.g., Hutchinson–Gilford progeria syndrome) and experimental studies suggest that changes in hemodynamic and mechanical forces are major modulators for vascular smooth muscle cell apoptosis. Furthermore, understanding the therapeutic effects of antihypertensive drugs related to apoptosis may identify pathways that can improve outcomes independent of the blood pressure fall. SummaryRegulation of vascular smooth muscle cell apoptosis is a potential target to modify pathological vascular remodeling and new drugs development.

Interleukin-18 and Macrophage Migration Inhibitory Factor Are Associated With Increased Carotid Intima–Media Thickening

Objective—Carotid intima–media thickening (IMT) is a form of vascular remodeling that has a strong genetic component. Recently, we discovered that in response to decreased carotid blood flow SJL mice developed the largest intima among 5 inbred strains. Because the SJL strain is prone to autoimmune diseases, we hypothesized that inflammation contributed to IMT in SJL mice. Methods and Results—We compared vascular remodeling (induced by 2 weeks of low flow) in 2 strains with small IMT (C3H/HeJ and C3HeB/FeJ) versus 2 strains with large IMT (FVB/NJ and SJL/J). Quantitative immunohistochemistry showed a dramatic increase in inflammatory cells per intima area in SJL compared with other strains. Microarray profiling of inflammatory gene mRNAs from carotids showed significant increases in interleukin (IL)-18 and Mif gene expression in SJL compared with C3HeB/FeJ mice. Increased expression of these genes was confirmed by quantitative reverse-transcription polymerase chain reaction and immunohistochemistry. Furthermore, greater cell proliferation in the intima of SJL accounted for increased intima–media thickening, whereas a higher level of apoptosis and a lower level of proliferation were observed in C3HeB/FeJ mice. Conclusion—The present study indicates that increased expression of Mif and IL-18 cytokines is associated with intima–media thickening in SJL mice, likely by stimulating inflammation and proliferation.