Hemodynamic Regulation of CD34+ Cell Localization and Differentiation in Experimental Aneurysms
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《动脉硬化血栓血管生物学》
From the Division of Vascular Surgery (E.S., M.S., R.L.D.), Stanford University, and Surgical Service, Veterans Affairs Palo Alto Health Care System, Palo Alto, Calif; and the Second Department of Pathology (H.N., K.K., H.M.), Akita University School of Medicine, Akita, Japan.
ABSTRACT
Objectives— Bone marrow-derived vascular progenitor cells (CD34+) are present in human and animal models of abdominal aortic aneurysm (AAA) disease. These preterminally differentiated cells may modulate disease resistance. We examined the influence of variable hemodynamic conditions on progenitor cell localization and differentiation in experimental AAAs.
Methods and Results— Murine AAAs were created via porcine pancreatic elastase (PPE) infusion. AAA blood flow was increased by aortocaval fistula (ACF) formation (HF-AAA), decreased via left iliac ligation (LF-AAA), or left unchanged (NF-AAA). ACF creation increased flow by 1700%, whereas iliac ligation decreased flow 79% compared with baseline (0.6±0.1 mL/min). Wall shear stress (WSS) increased or decreased accordingly, and remained elevated (9.2±2.0 dynes/cm2) in HF-AAA 14 days after PPE infusion. CD34+ cells were identified throughout the aortic wall in all flow conditions. Seven days after PPE infusion, HF-AAAs had more CD34+ cells than LF-AAA (187±10 versus 155±7 CD34+ cells/cross sectional, P<0.05), more medial smooth muscle cells, fewer infiltrative macrophages, and a smaller diameter than LF-AAA. LF-AAAs also contained more adventitial capillaries (CD34+ capillaries 181±12 versus 89±32/cross-sectional area in HF-AAA, P<0.05). The total progenitor cell/capillary index (CD34+ capillary plus CD31+ capillary/cross sectional area) was higher in LF-AAA (282±31 versus 129±47, P<0.05). Vascular endothelial (VEGF) and platelet-derived growth factor (PDGF) expression varied directly with capillary density between groups. Increased granulocyte-macrophage colony-stimulating factor (GM-CSF) expression was also present in LF-AAAs.
Conclusions— Hemodynamic conditions influence CD34+ cell localization and differentiation in experimental AAA. Adventitial capillary angiogenesis may augment inflammation and disease progression. Modulating cell lineage differentiation of mature progenitor cells may represent a novel therapeutic strategy to maintain medial cellularity and extracellular matrix integrity in AAA disease.
Vascular progenitor cells (CD34+ cells) localize within murine AAA, contributing to aneurysmal formation and progression. Luminal flow conditions regulate CD34+ cell differentiation and localization.
Key Words: blood flow ? wall shear stress ? vascular progenitor cell ? angiogenesis ? AAA ? smooth muscle cells ? macrophages
Introduction
Abdominal aortic aneurysm (AAA) is a highly lethal, age-related transmural degenerative disease of the distal aorta. Sentinel morphological events include aortic endothelial and medial smooth muscle cell apoptosis, medial elastic lamellar dissolution, and disorganized and ultimately ineffective compensatory extracellular matrix remodeling.1–4 These cellular and extracellular events presage progressive aortic diameter enlargement and eventual rupture.5 Specific biological mechanisms related to AAA initiation, growth, and eventual rupture remain speculative and highly controversial.6
Blood flow and wall shear stress (WSS) values vary along the length of the human aorta from the diaphragm to the aortic bifurcation. Compared with more proximal segments, the aneurysm-prone infrarenal aorta is chronically exposed to the highest peripheral resistance, lowest flow, and highest oscillatory WSS,7,8 conditions known to increase pro-inflammatory and pro-apoptotic gene expression in vitro and in vivo.9,10 The recent recognition of lower limb amputation and severe peripheral vascular disease as novel putative AAA risk factors highlight the potential pathogenic clinical significance of resistive aortic hemodynamic conditions.11,12
Variable flow conditions also alter the morphology and cellularity of experimental AAAs. Increased aortic flow reduces mural macrophage infiltration and increases luminal endothelial cell (EC) and medial smooth muscle cell (SMC) density while limiting AAA diameter.13,14 Bone marrow-derived circulating vascular progenitor cells (CD34+),15–18 recently identified in human AAAs,19 localize and proliferate within injured arteries20 and maturing vein grafts21 during mechano-transductive vascular remodeling. In this study, we investigated the influence of hemodynamic conditions on vascular progenitor cell localization and differentiation in experimental AAA.
Methods
Surgical and Anesthetic Preparation
All experimental surgical procedures were approved by the Institutional Animal Care and Use Committee of the Veterans Affairs Palo Alto Health Care System and were conducted in compliance with Stanford University Administrative Panel on Laboratory Animal Care Guidelines.
After intraperitoneal sodium pentobarbital injection (50 mg/kg), the abdominal aortae of wild-type 129/sv mice aged 8 to 10 weeks were isolated from the level of the left renal vein to the aortic bifurcation. After temporary ligature aortic control, a 30-gauge needle was advanced into the aortic bifurcation and withdrawn. Heat-tapered PE-10 tubing was advanced through the aortotomy and used to infuse 0.05 mL saline solution containing 15 U/mL type 1 porcine pancreatic elastase (PPE) (E-1250; Sigma) for 5 minutes. After infusion, the PE-10 tubing was withdrawn, the aortotomy closed with 10-0 suture, and aortic flow restored. Mice were recovered from surgery in separate cages with free access to food and water.
Creation and Measurement of Variable Flow AAA
High-flow (HF) AAAs were created by re-passage of the needle through the common wall between the aorta and inferior vena cava (aortocaval fistula creation) immediately before aortotomy closure (n=20). Left common iliac artery ligation was used to create low-flow (LF) AAAs (n=20). Normal-flow (NF) AAA mice underwent PPE infusion alone (n=20). ACF were also created without PPE infusion to serve as a HF control (ACF only, n=5). Aortae from mice without previous surgical manipulation were used as normal controls. Pre-infusion and postinfusion aortic flows were measured via ultrasonic flowmetry (Transonics Systems). WSS in dynes per square centimeter was calculated as: WSS (dyne/cm2)=4 x μ x BFR ÷ 60 x r3, where μ is the blood viscosity (0.035 poise),13,14 BFR is blood flow rate (mL/min), and r is aortic radius (cm). Maximum aortic diameter within the infused segment was measured pre-infusion, postinfusion, and at euthanization from 35-mm photomicrographs that included a reference scale.
Aortic Harvest and Preparation
Mice were euthanized via intentional anesthetic overdose at 1 hour and 1, 3, 7, and 14 days after PPE infusion. Final aortic diameter measurement was obtained immediately before euthanization. Pressure perfusion fixation was performed with 4% paraformaldehyde or 3% glutaraldehyde-buffered solution in 0.1 mol/L phosphate buffer (pH 7.4) infused via the left ventricle for 30 minutes at 20°C at 100 mm Hg. Representative aortae in each group were frozen in liquid nitrogen (LN2) for molecular analysis and saved at –80°C.
Aortic specimens for histological analysis were counter-stained with hematoxylin and eosin, as well as Masson trichrome stain. Specimens for scanning electron microscopy were dehydrated with absolute alcohol and dried using the critical point technique. After trimming, mounting, and coating with gold platinum, specimens were scanned using the JSM-5200 microscope (JEOL). Specimens for transmission electron microscopy were dehydrated and embedded in Epon. Semi-thin sections were examined to confirm proper cross-sectional orientation before ultra-thin sectioning for transmission electron microscopy. The sections were stained with lead citrate and uranyl acetate and imaged with a LEM2000 scanner (Topcon).
Histomorphometry
Histomorphometry was performed on Masson trichrome-stained histological sections as previously described.22 The length of the lumen and medial layers was obtained from direct measurement of aortic sections, and from these values AAA luminal diameter and cross-sectional area of aortic wall were calculated. A "shrinkage index" (x1.25 for length and x1.56 for area)22 was used to correct for distortion caused by fixation and staining procedures.
Qualitative mRNA Expression Analysis
Real-time polymerase chain reaction was performed using the GeneAmp 7700 sequence detection system (Applied Biosystems) as previously described.13,14 Briefly, high-quality total RNA was extracted using TRIzol reagent (GIBCO BRL) according to the manufacturer’s protocol. Total RNA was used to generate cDNA for oligo-dT oligodeoxynucleotide primer (T12–18) according to the manufacturer’s protocol (Superscript II reverse transcriptase; Invitrogen). The primers synthesized in Table I (available online at http://atvb.ahajournals.org) were designed using Primer Express software (Applied Biosystems). Equal amounts of cDNA were used in duplicate and amplified with the SYBR Green I Master mix system (Applied Biosystems). Thermal activation was initiated at 95°C for 10 minutes, followed by 40 cycles of polymerase chain reaction (melting for 15 seconds at 95°C and annealing/extension for 1 minute at 60°C). Amplification efficiencies were validated and normalized against ?-actin. Correct polymerase chain reaction product size was confirmed by electrophoresis through a 2% agarose gel with ethidium bromide.
Immunohistochemistry
Immunohistochemical staining was performed as previously described.13,14 Briefly, macrophages were identified by application of biotin rat antimouse Mac-2 primary antibody followed by purified streptavidin conjugated to horseradish peroxidase secondary antibody (Serotec). ECs were identified with goat antihuman polyclonal CD31 antibody (Santa Cruz Biotechnology) followed by biotinylated antigoat secondary antibody (Vector Laboratories). SMCs were identified with mouse monoclonal antismooth muscle -actin (SM- actin) antibody (Sigma) followed by biotinylated goat antimouse secondary antibody (Vector Laboratories). PBS was substituted for the primary antibody for the negative control. Endothelial and SMC proliferation were identified with monoclonal anti-BrdU antibody (Becton Dickinson) followed by biotinylated goat antimouse IgG (KPL Labs). PBS was again used as the primary antibody for negative control staining. Progenitor cells (CD34+ cells) were identified with monoclonal antibody against mouse CD34 (HyCult Biotechnology) followed by biotinylated goat antirat IgG (Serotec). The presence of CD34+ SMCs was confirmed with double staining; sections being finished with CD34 staining were subsequently incubated with monoclonal anti-SM- actin for 1 hour, followed by secondary antibody for 30 minutes as noted and ABC method according to the manufacturer’s protocol. Blue–gray color was developed by DAB kit (Vector Laboratories). SMCs stained blue–gray, and CD34+ cells stained brown.
Flow Cytometry Analysis
To quantify the number of circulating CD34+ cells, 1 mL blood was collected from HF-AAA, NF-AAA, LF-AAA, and ACF-only mice 5 days after PPE infusion. The red blood cells were lysed, and remaining cells were diluted to 1x107cells/mL. Spleen and bone marrow cells were also collected for positive control. Cells (1x106 in 100 μL) were incubated with monoclonal rat antimouse CD34 IgG2a antibody (HyCult Biotechnology) (1:100) at 4°C for 30 minutes followed by fluorescein isothiocyanate-conjugated antirat IgG (1:500) for 30 minutes. For negative control staining, normal rat serum was substituted for the primary antibody. The cells were analyzed by flow cytometry using a fluorescence-activated cell sorter Calibur (Becton-Dickinson) for at least 10 000 events.
Endothelial and Medial SMC Proliferation Indices
To investigate cell proliferation, selected mice at all time points received an intraperitoneal administration of BrdU at 50 mg/kg13 in physiological saline solution (5 mg/mL) 1 hour before termination to "pulse label" all cells entering DNA synthesis phase. BrdU-positive cells in the endothelium and media were counted on the entire cross-section (5 serial sections/case) as BrdU–EC/cross-section and BrdU–SMC/cross-section. All sections were counted twice and averaged for a final value.
CD34+ and CD31+ Cell Indices
Single CD34+ cells were counted on the entire cross-section (5 serial sections/case) as CD34+ cells/cross-section. CD34+ capillaries were counted on the entire cross-section as CD34+ capillaries/cross-section. CD31+ capillaries were counted on the entire cross-section as CD31+ capillaries/cross-section. All sections were counted twice and averaged for a final value.
Statistical Analysis
All data were expressed as mean±SD. Statistical analyses were performed via 1-way ANOVA with the Bonferroni/Dunn post-hoc correction for non-normally distributed populations. Differences were considered statistically significant at the P<0.05 level.
Results
ACF creation and iliac ligation were highly successful in varying aortic blood flow between groups (Figure 1A; immediate postoperative measurements). AAA instability precluded re-exposure for repeat flow measurements after the third day after PPE infusion. Therefore, late WSS calculations incorporated real-time diameter measurement and baseline post-ACF/iliac ligation/no-flow modification flow values in the HF/LF and NF groups, respectively.13,14 Resting baseline WSS in control aorta was 16.3±3.7 dynes/cm2. Although greatly increased initially in HF-AAA, calculated WSS declined progressively in all 3 groups as a function of AAA diameter enlargement (Figure 1B). Despite this decline, calculated WSS remained significantly higher in HF-AAA than NF-AAA or LF-AAA at all time points. PPE infusion immediately increased aortic diameter by 150% in HF-AAA, LF-AAA, and NF-AAA. Differences in flow and WSS began to further modify AAA progression from day 3 onward (Figure 1C). By day 14, AAA diameter was 3.2±0.1 mm in LF-AAA, 2.8±0.3 mm in NF-AAA, and 1.9±0.1 mm in HF-AAA (P<0.01 versus each other).
Figure 1. Blood flow, wall shear stress (WSS), and progression of variable flow AAAs. A, The influence of ACF creation and left iliac artery ligation on aortic flow (*P<0.01 versus NF-AAA, P<0.01 versus LF-AAA). B, WSS as a function of progressive AAA enlargement in variable flow AAA. C, Time course of AAA diameter progression as a function of luminal flow conditions.
Circulating CD34+ cells accounted for 0.01±0.003% of all peripheral blood cells in normal control mice, as compared with 0.09±0.02, 0.02±0.01, 0.03±0.01, and 0.06±0.01% for HF-AAA, NF-AAA, LF-AAA, and ACF-only aortae, respectively. Less than 5±1.4 CD34+ cells/cross-section were present in the adventitia of normal aortas. Creation of ACF alone did not increase this number significantly (data not shown). PPE infusion did dramatically increase mural CD34+ cells in all flow groups beginning on day 1 and peaking at day 7; the proportion of medial/total mural CD34+ cells decreased from 25% to 30% to 5% in all groups between 1 and 7 days after infusion. HF-AAA had more mural CD34+ cells/cross-sectional area than NF-AAA or LF-AAA at all time points studied (Table II, available online at http://atvb.ahajournals.org). Three morphologically distinct aortic CD34+ cell configurations were noted: (1) rounded cells grouped in clusters in the media and adventitia; (2) single spindle cells; and (3) adventitial capillary ECs (Figure 2A and 2B). Ultrastructural analysis confirmed the presence of morphological features consistent with progenitor cells: large nuclei, few mitochondria, and little endoplasmic reticulum (Figure IIG and IIH, available online at http://atvb.ahajournals.org).23,24 The presence of some CD34/-actin double-stained cells suggested that SMCs derived from CD34+ cells (Figure 2C and 2D).
Figure 2. CD34+ cells in HF-AAAs and LF-AAAs. Photomicrographs of CD34+ cells (brown stain) in LF-AAAs (A) and HF-AAAs (B). Single-spindle CD34+ cells are located in the media area (arrowheads). Several adventitial capillaries demonstrate CD34+ ECs (arrows). C and D, CD34/SM- actin double-staining (arrows, SM- actin+ cells stained dark gray) (original magnification x400).
As previously reported in rat models under longer infusion protocols,13 the PPE infusion precipitated significant endothelial denudation. Approximately 75% of ECs and 30% of SMCs were lost immediately after infusion. Reactive and restorative endothelial and SMC proliferation was observed from at least day 3 after PPE infusion; significantly more proliferation as determined by BrdU incorporation was noted in HF-AAA versus LF-AAA (Figure 3). More ECs per cross-luminal circumference were present in HF-AAA than NF-AAA or LF-AAA. EC density (as defined by the number of ECs/luminal circumferential length ) reached 49% of baseline by 7 days after PPE infusion in HF-AAA compared with only 18% in LF-AAA. Cross-sectional area of aortic wall (as an indirect measure of mural inflammation) was larger in LF-AAA than in HF-AAA, but SMC density (number of SMCs/cross-sectional area of aortic wall ) was less in LF-AAA than in HF-AAA (P<0.05 for all comparisons; Figure I, available online at http://atvb.ahajournals.org).
Figure 3. Endothelial and SMC proliferation in variable flow AAA. BrdU incorporation (ECs, arrows; SMCs, arrowheads) as a function of luminal flow conditions (A shows LF-AAA; B shows HF-AAA) (original magnification x400); C, BrdU+ EC cells per cross-section. D, BrdU + SMCs per cross-sectional area (CSA). *P<0.05 versus 3 days of PPE infusion; P<0.05 versus NF-AAA and HF-AAA; P<0.05 versus HF-AAA.
By scanning electron microscopy analysis, PPE infusion precipitated profound ECs loss with mural thrombus formation. EC proliferation, more evident in HF-AAA, was present from day 3. At 7 days, proliferating ECs almost completely covered the luminal surface of HF-AAA, whereas mural thrombi were still present in LF-AAA and NF-AAA. New SMCs with abundant mitochondria and rough endoplasmic reticulum were present in the aneurysm wall.
Mural macrophage infiltration was present from day 1 after PPE infusion and was highly flow-dependent; many more medial macrophages were present in LF-AAA compared with HF-AAA (Figure 4). Transmission electron microscopy confirmed the presence of macrophages and new medial and adventitial capillaries in all AAA flow groups by 3 days after PPE infusion. HF-AAA contained fewer CD34+ and CD34/31+ adventitial capillaries than NF-AAA and LF-AAA at 7 and 14 days, respectively (P<0.01; Table II).
Figure 4. Macrophage infiltration in variable flow AAAs. Macrophage staining (Mac-2, brown) shows extensive transmural infiltration in LF-AAA (A) and HF-AAA (B) by day 7 (original magnification x200). Transmission electron microscopy demonstrates subendothelial (arrowheads), medial, and adventitial (arrows) macrophage infiltration in AAA (C, D), as well as adventitial neocapillary (Cap) formation. IEL indicates internal elastic lamina.
Flow-dependent expression of relevant cell markers, cytokines, growth factors, and growth factor receptors were analyzed in a time-dependent fashion (Figure III, available online at http://atvb.ahajournals.org). LF-AAA demonstrated increased mural granulocyte-macrophage colony-stimulating factor (GM-CSF) expression at all time points compared with NF-AAA or HF-AAA. Endothelial adhesion molecule expression (CD31) was also relatively upregulated in LF-AAA. Expression of PDGF-A, PDGF-B, PDGF-C, and PDGF-D, as well as VEGF-A, VEGF-B, VEGF-C, and VEGF-D were all increased in LF-AAA as compared with HF-AAA, perhaps as a function of the increased numbers of capillaries present in the larger LF-AAA. VEGFR-1 and VEGFR-2 expression increased in parallel to VEGF expression.
Discussion
These experiments demonstrate the effect of flow conditions on vascular progenitor cell localization and differentiation in experimental AAA. Although rare in the intact healthy aortic wall, progenitor cells in evolving AAAs accumulate in the media and adventitia with the potential to differentiate into SMCs, macrophages, or capillaries. We also demonstrate for the first time to our knowledge that flow conditions mediate medial and adventitial capillary angiogenesis, an important correlate of mural inflammation.
Beyond recognition of selected cell surface markers, the precise phenotype of vascular progenitor cells in adult rodents and humans remains uncertain. Peripheral progenitor cells may be present in the arterial wall or in the circulation, but all are derived from and replenished by stem or progenitor cells of bone marrow origin.25 These cells may transdifferentiate into cardiomyocytes or endothelial or vascular SMCs, potentially playing a critical role in modulating arterial disease resistance and pathogenesis.26–30 In certain circumstances, bone marrow cells may also adopt the phenotype of mature cells by spontaneous cell fusion, as evidenced by the existence of over-diploid DNA content.31 Without quantifying the amount of DNA present in our "differentiated" CD34+ mesenchymal cells, we cannot exclude the possibility that some may have actually represented fused cells of bone marrow origin.
Differentiation of vascular progenitor cells into distinct lineages is directed in part via exposure to specific chemokines and growth factors such as VEGF and PDGF-BB. The VEGF family includes subtypes A, B, C, D, and E, all of which promote angiogenesis through interaction with the tyrosine receptor kinases VEGFR1 and VEGFR2. PDGF BB catalyzes outgrowth of -actin, myosin, and calponin (+) SMCs from human mononuclear CD34+ cells in culture.32 In a murine lung carcinoma model, tumor angiogenesis occurs via VEGF-induced chemo-attraction of CD34+ progenitor cells; differentiation into mature ECs occurs after exposure to angiopoietin-1 in tumor-conditioned medium.33 In the present study, VEGF, VEGFR, and PDGF were all upregulated during aneurysm progression and expansion. Increased VEGF/VEGFR expression was accompanied temporally by the development of large atypical adventitial neo-capillaries. This effect was tempered by flow conditions; LF-AAA demonstrated much more capillary angiogenesis and less medial SMC proliferation than HF-AAA. This suggests that in AAA disease, flow conditions directly or indirectly influence growth factor production that, in turn, mediates vascular progenitor cell localization and differentiation.
Mural angiogenesis or neovascularization may represent an important component of human AAA disease. Capillary angiogenesis or "neovascularization" is present in all 3 layers of the human AAA wall.34,35 This neovascularization is uniformly accompanied by inflammatory cell infiltration and fibroblast proliferation. Activated macrophages stimulate vascular proliferation,36 and macrophage-derived mediators promote all phases of angiogenesis.37–40 Neovascularization in turn may facilitate transmural macrophage infiltration, promoting mural elastolysis, a critical component of human AAA progression. In the present study, neovascularization, mural macrophage infiltration, and elastolysis all varied in inverse proportion to luminal flow. Whether neovascularization represents a stimulus or consequence of progressive mural inflammatory cell infiltration remains to be determined.
Progenitor cells may differentiate into macrophages at sites of arterial injury. Lineage determination may be manipulated by selective cell receptor inhibition; anti–c-fms antibody (macrophage differentiation inhibitor) promotes smooth muscle differentiation of localizing progenitor cells, whereas anti-PDGFR-? (SMC proliferation inhibitor) promotes macrophages differentiation.20 In vivo, GM-CSF similarly promotes differentiation of progenitor cells into macrophages and other myelomonocytic precursor cells, which in turn produce angiogenic factors that influence subsequent vascular progenitor cell differentiation and angiogenesis. In our models, AAA GM-CSF expression correlated with AAA diameter, mural macrophage density, and AAA progression. The possibility that cytokine-mediated in situ cellular differentiation may influence overall AAA inflammatory cell burden suggests to us a new way of potentially understanding the molecular mechanisms underlying AAA disease.
Local hemodynamic forces such as flow and WSS are important mediators of vascular remodeling in a variety of animal models.41–45 In normal arteries, increased flow and antegrade WSS promote arterial EC proliferation and migration and medial SMC proliferation resulting in adaptive enlargement and luminal tortuosity. In rat AAA models, HF and shear stress promote EC proliferation, luminal resurfacing, mural SMC proliferation, and prevent transmural macrophage infiltration while limiting AAA progression. Reduced flow, by comparison, promotes AAA progression. The current study results provide reassurance that these previously reported relationships are not species-specific. Additional experiments are ongoing to validate the relationships between flow conditions and disease progression in complementary models (eg, murine angiotensin II/apolipoprotein E–/– and CaCl2).46
HF aortic conditions maintain SMC density as functions of increased proliferation and reduced apoptosis. Increasing SMC density without modifying luminal flow conditions is also highly effective in limiting experimental AAA progression. Luminal seeding of syngenic SMCs in a xenograft aortic transplant AAA model attenuates elastin degradation, decreases monocyte and macrophage infiltration, and limits AAA progression despite ongoing proteolytic enzyme expression and activation from seeded cells.47 Luminal seeding reduces transmural inflammation and proteolysis, suggesting that hemodynamic influences mediate production and release of diffusible anti-inflammatory paracrine mediators in mature SMCs in addition to flow effects on circulating progenitor cells. Although the identity of these mediators remains uncertain, they may also include growth or chemotactic factors. Delivery of basic fibroblast growth factor in expression plasmids to aortic SMCs via electroporation increases SMC proliferation, reduces AAA diameter, and downregulates proteolytic enzyme expression in the PPE infusion AAA model.48 Progenitor cells are innately resistant to pro-aneurysmal environmental stresses such as reactive oxygen species production,49 an important pathogenic feature of experimental and human AAA disease that contributes to SMC apoptosis and cell loss,50,51 and identifying and characterizing mechanisms that induce progenitor cell recruitment and SMC differentiation may translate into increased medial retention and reduced disease progression.
In summary, luminal hemodynamic conditions modulate murine experimental AAA progression and influence localization and cell lineage differentiation of adult vascular progenitor cells. LF conditions stimulate medial–adventitial angiogenesis, transmural inflammatory cell infiltration, and aneurysm progression. HF conditions augment medial smooth muscle cellularity and limit aneurysm progression. Directing progenitor cells differentiation into disease-resistant cell lineages may represents a new therapeutic alternative for treatment or prevention of AAA disease.
Acknowledgments
Supported was provided in part by the National Heart, Lung, and Blood Institute (RO1 HL46338), the Palo Alto Institute for Research and Education, and the Japanese Ministry of Education, Culture, Sports, and Science (grant 16300200).
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ABSTRACT
Objectives— Bone marrow-derived vascular progenitor cells (CD34+) are present in human and animal models of abdominal aortic aneurysm (AAA) disease. These preterminally differentiated cells may modulate disease resistance. We examined the influence of variable hemodynamic conditions on progenitor cell localization and differentiation in experimental AAAs.
Methods and Results— Murine AAAs were created via porcine pancreatic elastase (PPE) infusion. AAA blood flow was increased by aortocaval fistula (ACF) formation (HF-AAA), decreased via left iliac ligation (LF-AAA), or left unchanged (NF-AAA). ACF creation increased flow by 1700%, whereas iliac ligation decreased flow 79% compared with baseline (0.6±0.1 mL/min). Wall shear stress (WSS) increased or decreased accordingly, and remained elevated (9.2±2.0 dynes/cm2) in HF-AAA 14 days after PPE infusion. CD34+ cells were identified throughout the aortic wall in all flow conditions. Seven days after PPE infusion, HF-AAAs had more CD34+ cells than LF-AAA (187±10 versus 155±7 CD34+ cells/cross sectional, P<0.05), more medial smooth muscle cells, fewer infiltrative macrophages, and a smaller diameter than LF-AAA. LF-AAAs also contained more adventitial capillaries (CD34+ capillaries 181±12 versus 89±32/cross-sectional area in HF-AAA, P<0.05). The total progenitor cell/capillary index (CD34+ capillary plus CD31+ capillary/cross sectional area) was higher in LF-AAA (282±31 versus 129±47, P<0.05). Vascular endothelial (VEGF) and platelet-derived growth factor (PDGF) expression varied directly with capillary density between groups. Increased granulocyte-macrophage colony-stimulating factor (GM-CSF) expression was also present in LF-AAAs.
Conclusions— Hemodynamic conditions influence CD34+ cell localization and differentiation in experimental AAA. Adventitial capillary angiogenesis may augment inflammation and disease progression. Modulating cell lineage differentiation of mature progenitor cells may represent a novel therapeutic strategy to maintain medial cellularity and extracellular matrix integrity in AAA disease.
Vascular progenitor cells (CD34+ cells) localize within murine AAA, contributing to aneurysmal formation and progression. Luminal flow conditions regulate CD34+ cell differentiation and localization.
Key Words: blood flow ? wall shear stress ? vascular progenitor cell ? angiogenesis ? AAA ? smooth muscle cells ? macrophages
Introduction
Abdominal aortic aneurysm (AAA) is a highly lethal, age-related transmural degenerative disease of the distal aorta. Sentinel morphological events include aortic endothelial and medial smooth muscle cell apoptosis, medial elastic lamellar dissolution, and disorganized and ultimately ineffective compensatory extracellular matrix remodeling.1–4 These cellular and extracellular events presage progressive aortic diameter enlargement and eventual rupture.5 Specific biological mechanisms related to AAA initiation, growth, and eventual rupture remain speculative and highly controversial.6
Blood flow and wall shear stress (WSS) values vary along the length of the human aorta from the diaphragm to the aortic bifurcation. Compared with more proximal segments, the aneurysm-prone infrarenal aorta is chronically exposed to the highest peripheral resistance, lowest flow, and highest oscillatory WSS,7,8 conditions known to increase pro-inflammatory and pro-apoptotic gene expression in vitro and in vivo.9,10 The recent recognition of lower limb amputation and severe peripheral vascular disease as novel putative AAA risk factors highlight the potential pathogenic clinical significance of resistive aortic hemodynamic conditions.11,12
Variable flow conditions also alter the morphology and cellularity of experimental AAAs. Increased aortic flow reduces mural macrophage infiltration and increases luminal endothelial cell (EC) and medial smooth muscle cell (SMC) density while limiting AAA diameter.13,14 Bone marrow-derived circulating vascular progenitor cells (CD34+),15–18 recently identified in human AAAs,19 localize and proliferate within injured arteries20 and maturing vein grafts21 during mechano-transductive vascular remodeling. In this study, we investigated the influence of hemodynamic conditions on vascular progenitor cell localization and differentiation in experimental AAA.
Methods
Surgical and Anesthetic Preparation
All experimental surgical procedures were approved by the Institutional Animal Care and Use Committee of the Veterans Affairs Palo Alto Health Care System and were conducted in compliance with Stanford University Administrative Panel on Laboratory Animal Care Guidelines.
After intraperitoneal sodium pentobarbital injection (50 mg/kg), the abdominal aortae of wild-type 129/sv mice aged 8 to 10 weeks were isolated from the level of the left renal vein to the aortic bifurcation. After temporary ligature aortic control, a 30-gauge needle was advanced into the aortic bifurcation and withdrawn. Heat-tapered PE-10 tubing was advanced through the aortotomy and used to infuse 0.05 mL saline solution containing 15 U/mL type 1 porcine pancreatic elastase (PPE) (E-1250; Sigma) for 5 minutes. After infusion, the PE-10 tubing was withdrawn, the aortotomy closed with 10-0 suture, and aortic flow restored. Mice were recovered from surgery in separate cages with free access to food and water.
Creation and Measurement of Variable Flow AAA
High-flow (HF) AAAs were created by re-passage of the needle through the common wall between the aorta and inferior vena cava (aortocaval fistula creation) immediately before aortotomy closure (n=20). Left common iliac artery ligation was used to create low-flow (LF) AAAs (n=20). Normal-flow (NF) AAA mice underwent PPE infusion alone (n=20). ACF were also created without PPE infusion to serve as a HF control (ACF only, n=5). Aortae from mice without previous surgical manipulation were used as normal controls. Pre-infusion and postinfusion aortic flows were measured via ultrasonic flowmetry (Transonics Systems). WSS in dynes per square centimeter was calculated as: WSS (dyne/cm2)=4 x μ x BFR ÷ 60 x r3, where μ is the blood viscosity (0.035 poise),13,14 BFR is blood flow rate (mL/min), and r is aortic radius (cm). Maximum aortic diameter within the infused segment was measured pre-infusion, postinfusion, and at euthanization from 35-mm photomicrographs that included a reference scale.
Aortic Harvest and Preparation
Mice were euthanized via intentional anesthetic overdose at 1 hour and 1, 3, 7, and 14 days after PPE infusion. Final aortic diameter measurement was obtained immediately before euthanization. Pressure perfusion fixation was performed with 4% paraformaldehyde or 3% glutaraldehyde-buffered solution in 0.1 mol/L phosphate buffer (pH 7.4) infused via the left ventricle for 30 minutes at 20°C at 100 mm Hg. Representative aortae in each group were frozen in liquid nitrogen (LN2) for molecular analysis and saved at –80°C.
Aortic specimens for histological analysis were counter-stained with hematoxylin and eosin, as well as Masson trichrome stain. Specimens for scanning electron microscopy were dehydrated with absolute alcohol and dried using the critical point technique. After trimming, mounting, and coating with gold platinum, specimens were scanned using the JSM-5200 microscope (JEOL). Specimens for transmission electron microscopy were dehydrated and embedded in Epon. Semi-thin sections were examined to confirm proper cross-sectional orientation before ultra-thin sectioning for transmission electron microscopy. The sections were stained with lead citrate and uranyl acetate and imaged with a LEM2000 scanner (Topcon).
Histomorphometry
Histomorphometry was performed on Masson trichrome-stained histological sections as previously described.22 The length of the lumen and medial layers was obtained from direct measurement of aortic sections, and from these values AAA luminal diameter and cross-sectional area of aortic wall were calculated. A "shrinkage index" (x1.25 for length and x1.56 for area)22 was used to correct for distortion caused by fixation and staining procedures.
Qualitative mRNA Expression Analysis
Real-time polymerase chain reaction was performed using the GeneAmp 7700 sequence detection system (Applied Biosystems) as previously described.13,14 Briefly, high-quality total RNA was extracted using TRIzol reagent (GIBCO BRL) according to the manufacturer’s protocol. Total RNA was used to generate cDNA for oligo-dT oligodeoxynucleotide primer (T12–18) according to the manufacturer’s protocol (Superscript II reverse transcriptase; Invitrogen). The primers synthesized in Table I (available online at http://atvb.ahajournals.org) were designed using Primer Express software (Applied Biosystems). Equal amounts of cDNA were used in duplicate and amplified with the SYBR Green I Master mix system (Applied Biosystems). Thermal activation was initiated at 95°C for 10 minutes, followed by 40 cycles of polymerase chain reaction (melting for 15 seconds at 95°C and annealing/extension for 1 minute at 60°C). Amplification efficiencies were validated and normalized against ?-actin. Correct polymerase chain reaction product size was confirmed by electrophoresis through a 2% agarose gel with ethidium bromide.
Immunohistochemistry
Immunohistochemical staining was performed as previously described.13,14 Briefly, macrophages were identified by application of biotin rat antimouse Mac-2 primary antibody followed by purified streptavidin conjugated to horseradish peroxidase secondary antibody (Serotec). ECs were identified with goat antihuman polyclonal CD31 antibody (Santa Cruz Biotechnology) followed by biotinylated antigoat secondary antibody (Vector Laboratories). SMCs were identified with mouse monoclonal antismooth muscle -actin (SM- actin) antibody (Sigma) followed by biotinylated goat antimouse secondary antibody (Vector Laboratories). PBS was substituted for the primary antibody for the negative control. Endothelial and SMC proliferation were identified with monoclonal anti-BrdU antibody (Becton Dickinson) followed by biotinylated goat antimouse IgG (KPL Labs). PBS was again used as the primary antibody for negative control staining. Progenitor cells (CD34+ cells) were identified with monoclonal antibody against mouse CD34 (HyCult Biotechnology) followed by biotinylated goat antirat IgG (Serotec). The presence of CD34+ SMCs was confirmed with double staining; sections being finished with CD34 staining were subsequently incubated with monoclonal anti-SM- actin for 1 hour, followed by secondary antibody for 30 minutes as noted and ABC method according to the manufacturer’s protocol. Blue–gray color was developed by DAB kit (Vector Laboratories). SMCs stained blue–gray, and CD34+ cells stained brown.
Flow Cytometry Analysis
To quantify the number of circulating CD34+ cells, 1 mL blood was collected from HF-AAA, NF-AAA, LF-AAA, and ACF-only mice 5 days after PPE infusion. The red blood cells were lysed, and remaining cells were diluted to 1x107cells/mL. Spleen and bone marrow cells were also collected for positive control. Cells (1x106 in 100 μL) were incubated with monoclonal rat antimouse CD34 IgG2a antibody (HyCult Biotechnology) (1:100) at 4°C for 30 minutes followed by fluorescein isothiocyanate-conjugated antirat IgG (1:500) for 30 minutes. For negative control staining, normal rat serum was substituted for the primary antibody. The cells were analyzed by flow cytometry using a fluorescence-activated cell sorter Calibur (Becton-Dickinson) for at least 10 000 events.
Endothelial and Medial SMC Proliferation Indices
To investigate cell proliferation, selected mice at all time points received an intraperitoneal administration of BrdU at 50 mg/kg13 in physiological saline solution (5 mg/mL) 1 hour before termination to "pulse label" all cells entering DNA synthesis phase. BrdU-positive cells in the endothelium and media were counted on the entire cross-section (5 serial sections/case) as BrdU–EC/cross-section and BrdU–SMC/cross-section. All sections were counted twice and averaged for a final value.
CD34+ and CD31+ Cell Indices
Single CD34+ cells were counted on the entire cross-section (5 serial sections/case) as CD34+ cells/cross-section. CD34+ capillaries were counted on the entire cross-section as CD34+ capillaries/cross-section. CD31+ capillaries were counted on the entire cross-section as CD31+ capillaries/cross-section. All sections were counted twice and averaged for a final value.
Statistical Analysis
All data were expressed as mean±SD. Statistical analyses were performed via 1-way ANOVA with the Bonferroni/Dunn post-hoc correction for non-normally distributed populations. Differences were considered statistically significant at the P<0.05 level.
Results
ACF creation and iliac ligation were highly successful in varying aortic blood flow between groups (Figure 1A; immediate postoperative measurements). AAA instability precluded re-exposure for repeat flow measurements after the third day after PPE infusion. Therefore, late WSS calculations incorporated real-time diameter measurement and baseline post-ACF/iliac ligation/no-flow modification flow values in the HF/LF and NF groups, respectively.13,14 Resting baseline WSS in control aorta was 16.3±3.7 dynes/cm2. Although greatly increased initially in HF-AAA, calculated WSS declined progressively in all 3 groups as a function of AAA diameter enlargement (Figure 1B). Despite this decline, calculated WSS remained significantly higher in HF-AAA than NF-AAA or LF-AAA at all time points. PPE infusion immediately increased aortic diameter by 150% in HF-AAA, LF-AAA, and NF-AAA. Differences in flow and WSS began to further modify AAA progression from day 3 onward (Figure 1C). By day 14, AAA diameter was 3.2±0.1 mm in LF-AAA, 2.8±0.3 mm in NF-AAA, and 1.9±0.1 mm in HF-AAA (P<0.01 versus each other).
Figure 1. Blood flow, wall shear stress (WSS), and progression of variable flow AAAs. A, The influence of ACF creation and left iliac artery ligation on aortic flow (*P<0.01 versus NF-AAA, P<0.01 versus LF-AAA). B, WSS as a function of progressive AAA enlargement in variable flow AAA. C, Time course of AAA diameter progression as a function of luminal flow conditions.
Circulating CD34+ cells accounted for 0.01±0.003% of all peripheral blood cells in normal control mice, as compared with 0.09±0.02, 0.02±0.01, 0.03±0.01, and 0.06±0.01% for HF-AAA, NF-AAA, LF-AAA, and ACF-only aortae, respectively. Less than 5±1.4 CD34+ cells/cross-section were present in the adventitia of normal aortas. Creation of ACF alone did not increase this number significantly (data not shown). PPE infusion did dramatically increase mural CD34+ cells in all flow groups beginning on day 1 and peaking at day 7; the proportion of medial/total mural CD34+ cells decreased from 25% to 30% to 5% in all groups between 1 and 7 days after infusion. HF-AAA had more mural CD34+ cells/cross-sectional area than NF-AAA or LF-AAA at all time points studied (Table II, available online at http://atvb.ahajournals.org). Three morphologically distinct aortic CD34+ cell configurations were noted: (1) rounded cells grouped in clusters in the media and adventitia; (2) single spindle cells; and (3) adventitial capillary ECs (Figure 2A and 2B). Ultrastructural analysis confirmed the presence of morphological features consistent with progenitor cells: large nuclei, few mitochondria, and little endoplasmic reticulum (Figure IIG and IIH, available online at http://atvb.ahajournals.org).23,24 The presence of some CD34/-actin double-stained cells suggested that SMCs derived from CD34+ cells (Figure 2C and 2D).
Figure 2. CD34+ cells in HF-AAAs and LF-AAAs. Photomicrographs of CD34+ cells (brown stain) in LF-AAAs (A) and HF-AAAs (B). Single-spindle CD34+ cells are located in the media area (arrowheads). Several adventitial capillaries demonstrate CD34+ ECs (arrows). C and D, CD34/SM- actin double-staining (arrows, SM- actin+ cells stained dark gray) (original magnification x400).
As previously reported in rat models under longer infusion protocols,13 the PPE infusion precipitated significant endothelial denudation. Approximately 75% of ECs and 30% of SMCs were lost immediately after infusion. Reactive and restorative endothelial and SMC proliferation was observed from at least day 3 after PPE infusion; significantly more proliferation as determined by BrdU incorporation was noted in HF-AAA versus LF-AAA (Figure 3). More ECs per cross-luminal circumference were present in HF-AAA than NF-AAA or LF-AAA. EC density (as defined by the number of ECs/luminal circumferential length ) reached 49% of baseline by 7 days after PPE infusion in HF-AAA compared with only 18% in LF-AAA. Cross-sectional area of aortic wall (as an indirect measure of mural inflammation) was larger in LF-AAA than in HF-AAA, but SMC density (number of SMCs/cross-sectional area of aortic wall ) was less in LF-AAA than in HF-AAA (P<0.05 for all comparisons; Figure I, available online at http://atvb.ahajournals.org).
Figure 3. Endothelial and SMC proliferation in variable flow AAA. BrdU incorporation (ECs, arrows; SMCs, arrowheads) as a function of luminal flow conditions (A shows LF-AAA; B shows HF-AAA) (original magnification x400); C, BrdU+ EC cells per cross-section. D, BrdU + SMCs per cross-sectional area (CSA). *P<0.05 versus 3 days of PPE infusion; P<0.05 versus NF-AAA and HF-AAA; P<0.05 versus HF-AAA.
By scanning electron microscopy analysis, PPE infusion precipitated profound ECs loss with mural thrombus formation. EC proliferation, more evident in HF-AAA, was present from day 3. At 7 days, proliferating ECs almost completely covered the luminal surface of HF-AAA, whereas mural thrombi were still present in LF-AAA and NF-AAA. New SMCs with abundant mitochondria and rough endoplasmic reticulum were present in the aneurysm wall.
Mural macrophage infiltration was present from day 1 after PPE infusion and was highly flow-dependent; many more medial macrophages were present in LF-AAA compared with HF-AAA (Figure 4). Transmission electron microscopy confirmed the presence of macrophages and new medial and adventitial capillaries in all AAA flow groups by 3 days after PPE infusion. HF-AAA contained fewer CD34+ and CD34/31+ adventitial capillaries than NF-AAA and LF-AAA at 7 and 14 days, respectively (P<0.01; Table II).
Figure 4. Macrophage infiltration in variable flow AAAs. Macrophage staining (Mac-2, brown) shows extensive transmural infiltration in LF-AAA (A) and HF-AAA (B) by day 7 (original magnification x200). Transmission electron microscopy demonstrates subendothelial (arrowheads), medial, and adventitial (arrows) macrophage infiltration in AAA (C, D), as well as adventitial neocapillary (Cap) formation. IEL indicates internal elastic lamina.
Flow-dependent expression of relevant cell markers, cytokines, growth factors, and growth factor receptors were analyzed in a time-dependent fashion (Figure III, available online at http://atvb.ahajournals.org). LF-AAA demonstrated increased mural granulocyte-macrophage colony-stimulating factor (GM-CSF) expression at all time points compared with NF-AAA or HF-AAA. Endothelial adhesion molecule expression (CD31) was also relatively upregulated in LF-AAA. Expression of PDGF-A, PDGF-B, PDGF-C, and PDGF-D, as well as VEGF-A, VEGF-B, VEGF-C, and VEGF-D were all increased in LF-AAA as compared with HF-AAA, perhaps as a function of the increased numbers of capillaries present in the larger LF-AAA. VEGFR-1 and VEGFR-2 expression increased in parallel to VEGF expression.
Discussion
These experiments demonstrate the effect of flow conditions on vascular progenitor cell localization and differentiation in experimental AAA. Although rare in the intact healthy aortic wall, progenitor cells in evolving AAAs accumulate in the media and adventitia with the potential to differentiate into SMCs, macrophages, or capillaries. We also demonstrate for the first time to our knowledge that flow conditions mediate medial and adventitial capillary angiogenesis, an important correlate of mural inflammation.
Beyond recognition of selected cell surface markers, the precise phenotype of vascular progenitor cells in adult rodents and humans remains uncertain. Peripheral progenitor cells may be present in the arterial wall or in the circulation, but all are derived from and replenished by stem or progenitor cells of bone marrow origin.25 These cells may transdifferentiate into cardiomyocytes or endothelial or vascular SMCs, potentially playing a critical role in modulating arterial disease resistance and pathogenesis.26–30 In certain circumstances, bone marrow cells may also adopt the phenotype of mature cells by spontaneous cell fusion, as evidenced by the existence of over-diploid DNA content.31 Without quantifying the amount of DNA present in our "differentiated" CD34+ mesenchymal cells, we cannot exclude the possibility that some may have actually represented fused cells of bone marrow origin.
Differentiation of vascular progenitor cells into distinct lineages is directed in part via exposure to specific chemokines and growth factors such as VEGF and PDGF-BB. The VEGF family includes subtypes A, B, C, D, and E, all of which promote angiogenesis through interaction with the tyrosine receptor kinases VEGFR1 and VEGFR2. PDGF BB catalyzes outgrowth of -actin, myosin, and calponin (+) SMCs from human mononuclear CD34+ cells in culture.32 In a murine lung carcinoma model, tumor angiogenesis occurs via VEGF-induced chemo-attraction of CD34+ progenitor cells; differentiation into mature ECs occurs after exposure to angiopoietin-1 in tumor-conditioned medium.33 In the present study, VEGF, VEGFR, and PDGF were all upregulated during aneurysm progression and expansion. Increased VEGF/VEGFR expression was accompanied temporally by the development of large atypical adventitial neo-capillaries. This effect was tempered by flow conditions; LF-AAA demonstrated much more capillary angiogenesis and less medial SMC proliferation than HF-AAA. This suggests that in AAA disease, flow conditions directly or indirectly influence growth factor production that, in turn, mediates vascular progenitor cell localization and differentiation.
Mural angiogenesis or neovascularization may represent an important component of human AAA disease. Capillary angiogenesis or "neovascularization" is present in all 3 layers of the human AAA wall.34,35 This neovascularization is uniformly accompanied by inflammatory cell infiltration and fibroblast proliferation. Activated macrophages stimulate vascular proliferation,36 and macrophage-derived mediators promote all phases of angiogenesis.37–40 Neovascularization in turn may facilitate transmural macrophage infiltration, promoting mural elastolysis, a critical component of human AAA progression. In the present study, neovascularization, mural macrophage infiltration, and elastolysis all varied in inverse proportion to luminal flow. Whether neovascularization represents a stimulus or consequence of progressive mural inflammatory cell infiltration remains to be determined.
Progenitor cells may differentiate into macrophages at sites of arterial injury. Lineage determination may be manipulated by selective cell receptor inhibition; anti–c-fms antibody (macrophage differentiation inhibitor) promotes smooth muscle differentiation of localizing progenitor cells, whereas anti-PDGFR-? (SMC proliferation inhibitor) promotes macrophages differentiation.20 In vivo, GM-CSF similarly promotes differentiation of progenitor cells into macrophages and other myelomonocytic precursor cells, which in turn produce angiogenic factors that influence subsequent vascular progenitor cell differentiation and angiogenesis. In our models, AAA GM-CSF expression correlated with AAA diameter, mural macrophage density, and AAA progression. The possibility that cytokine-mediated in situ cellular differentiation may influence overall AAA inflammatory cell burden suggests to us a new way of potentially understanding the molecular mechanisms underlying AAA disease.
Local hemodynamic forces such as flow and WSS are important mediators of vascular remodeling in a variety of animal models.41–45 In normal arteries, increased flow and antegrade WSS promote arterial EC proliferation and migration and medial SMC proliferation resulting in adaptive enlargement and luminal tortuosity. In rat AAA models, HF and shear stress promote EC proliferation, luminal resurfacing, mural SMC proliferation, and prevent transmural macrophage infiltration while limiting AAA progression. Reduced flow, by comparison, promotes AAA progression. The current study results provide reassurance that these previously reported relationships are not species-specific. Additional experiments are ongoing to validate the relationships between flow conditions and disease progression in complementary models (eg, murine angiotensin II/apolipoprotein E–/– and CaCl2).46
HF aortic conditions maintain SMC density as functions of increased proliferation and reduced apoptosis. Increasing SMC density without modifying luminal flow conditions is also highly effective in limiting experimental AAA progression. Luminal seeding of syngenic SMCs in a xenograft aortic transplant AAA model attenuates elastin degradation, decreases monocyte and macrophage infiltration, and limits AAA progression despite ongoing proteolytic enzyme expression and activation from seeded cells.47 Luminal seeding reduces transmural inflammation and proteolysis, suggesting that hemodynamic influences mediate production and release of diffusible anti-inflammatory paracrine mediators in mature SMCs in addition to flow effects on circulating progenitor cells. Although the identity of these mediators remains uncertain, they may also include growth or chemotactic factors. Delivery of basic fibroblast growth factor in expression plasmids to aortic SMCs via electroporation increases SMC proliferation, reduces AAA diameter, and downregulates proteolytic enzyme expression in the PPE infusion AAA model.48 Progenitor cells are innately resistant to pro-aneurysmal environmental stresses such as reactive oxygen species production,49 an important pathogenic feature of experimental and human AAA disease that contributes to SMC apoptosis and cell loss,50,51 and identifying and characterizing mechanisms that induce progenitor cell recruitment and SMC differentiation may translate into increased medial retention and reduced disease progression.
In summary, luminal hemodynamic conditions modulate murine experimental AAA progression and influence localization and cell lineage differentiation of adult vascular progenitor cells. LF conditions stimulate medial–adventitial angiogenesis, transmural inflammatory cell infiltration, and aneurysm progression. HF conditions augment medial smooth muscle cellularity and limit aneurysm progression. Directing progenitor cells differentiation into disease-resistant cell lineages may represents a new therapeutic alternative for treatment or prevention of AAA disease.
Acknowledgments
Supported was provided in part by the National Heart, Lung, and Blood Institute (RO1 HL46338), the Palo Alto Institute for Research and Education, and the Japanese Ministry of Education, Culture, Sports, and Science (grant 16300200).
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