Nestin identifies a subpopulation of rat ventricular fibroblasts and participates in cell migration.

Fibroblast progenitor cells migrate to the endocardial region during cardiogenesis, and the migration of ventricular fibroblasts to the ischemically damaged region of the infarcted adult heart is a seminal event of reparative fibrosis. The intermediate filament protein nestin is implicated in cell migration and expression identified in a subpopulation of scar-derived myofibroblasts. The present study tested the hypothesis that fibroblast progenitor cells express nestin, and the intermediate filament protein drives the migratory phenotype of ventricular fibroblasts. Transcription factor 21 (Tcf21)- and Wilms tumor 1 (WT1)-fibroblast progenitor cells identified in the epicardial/endocardial regions of the E12.5- to E13.5-day embryonic mouse heart predominantly expressed nestin. Nuclear Tcf21/WT1 staining was identified in neonatal rat ventricular fibroblasts (NNVFbs), and a subpopulation coexpressed nestin. Nuclear Tcf21/WT1 expression persisted in adult rat ventricular fibroblasts, whereas nestin protein levels were downregulated. Nestin-expressing NNVFbs exhibited a unique phenotype as the subpopulation was refractory to cell cycle reentry in response to selective stimuli. Nestin(-)- and nestin(+)-scar-derived rat myofibroblasts plated in Matrigel unmasked a migratory phenotype characterized by the de novo formation of lumen-like structures. The elongated membrane projections emanating from scar myofibroblasts delineating the boundary of lumen-like structures expressed nestin. Lentiviral short-hairpin RNA (shRNA)-mediated nestin depletion inhibited the in vitro migratory response of NNVFbs as the wound radius was significantly larger compared with NNVFbs infected with the empty lentivirus. Thus, nestin represents a marker of embryonic Tcf21/WT1(+)-fibroblast progenitor cells. The neonatal rat heart contains a distinct subpopulation of nestin-immunoreactive Tcf21/WT1(+) fibroblasts refractory to cell cycle reentry, and the intermediate filament protein may preferentially facilitate ventricular fibroblast migration during physiological/pathological remodeling.NEW & NOTEWORTHY Tcf21/WT1(+)-fibroblast progenitor cells of the embryonic mouse heart predominantly express the intermediate filament protein nestin. A subpopulation of Tcf21/WT1(+)-neonatal rat ventricular fibroblasts express nestin and are refractory to selective stimuli influencing cell cycle reentry. Scar-derived myofibroblasts plated in Matrigel elicit the formation of lumen-like structures characterized by the appearance of nestin(+)-membrane projections. Lentiviral shRNA-mediated nestin depletion in a subpopulation of neonatal rat ventricular fibroblasts suppressed the migratory response following the in vitro scratch assay.

Structural dysregulation of the pulmonary autograft was associated with a greater density of p16INK4A-vascular smooth muscle cells.

The present study tested the hypothesis that a senescent phenotype of vascular smooth muscle cells (VSMCs) may represent the seminal event linked to maladaptive pulmonary autograft remodeling of a small number of patients that underwent the Ross procedure. The diameter of the pulmonary autograft (47±4 mm) of three male patients was significantly greater compared to the pulmonary artery (26±1 mm) excised from bicuspid aortic valve (BAV) patients. The pulmonary autograft was associated with a neointimal region and the adjacent medial region was significantly thinner compared to the pulmonary artery of BAV patients. Structural dysregulation was evident as elastin content of the medial region was significantly reduced in the pulmonary autograft compared to the pulmonary artery of BAV patients. By contrast, collagen content of the medial region of the pulmonary autograft and the pulmonary artery of BAV patients was not significantly different. Reduced medial elastin content of the pulmonary autograft was associated with increased protein levels of matrix metalloproteinase-9. The latter phenotype was not attributed to a robust inflammatory response as the percentage of Mac-2(+)-infiltrating monocytes/macrophages was similar between groups. A senescent phenotype was identified as protein levels of the cell cycle inhibitor p27kip1 were upregulated and the density of p16INK4A/non-muscle myosin IIB(+)-VSMCs was significantly greater in the pulmonary autograft compared to the pulmonary artery of BAV patients. Thus, senescent VSMCs may represent the predominant cellular source of increased matrix metalloproteinase-9 protein expression translating to maladaptive pulmonary autograft remodeling characterized by elastin degradation, medial thinning and neointimal formation.

The ascending aorta of male hypertensive bicuspid aortic valve patients preferentially associated with a cellular aneurysmal phenotype.

Male sex and hypertension represent risk factors in the progression of an aortic aneurysm. The present study examined the morphological/cellular phenotype of the ascending aorta (AA) of male and female patients diagnosed with a bicuspid aortic valve (BAV) to test the hypothesis that hypertension-induced remodeling of male BAV patients preferentially recapitulated the expression of a panel of proteins favoring aneurysm formation. The diameter of the AA of hypertensive male (35 ± 6 mm) and female (39 ± 5 mm) BAV patients was comparable to normotensive patients reflecting an early phase of vessel expansion. Morphological/structural remodeling of the medial region of the AA of male normotensive and hypertensive BAV patients were comparable. Protein levels of non-muscle myosin IIB, the cell cycle inhibitor p27kip1, tumor suppressor p53 and matrix metalloproteinase-2 and -9 were significantly upregulated in the AA of male hypertensive BAV patients. In female hypertensive BAV patients, collagen content was significantly increased whereas elastin content and medial width of the AA were similar to normotensive BAV patients. In the AA of female hypertensive BAV patients, matrix metalloproteinase-9 and p27kip1 protein levels were unchanged whereas p53 and matrix metalloproteinase-2 protein expression was significantly reduced. Nestin protein levels were diminished in the AA of male and female hypertensive BAV patients. Thus, sexual dimorphic remodeling of the AA was prevalent in hypertensive BAV patients. Moreover, during the early phase of vessel expansion, the AA of male hypertensive BAV patients was preferentially associated with the upregulation of a panel of proteins linked to progressive dilatation and potential aneurysm formation.

Distinct Expression of Nonmuscle Myosin IIB in Pulmonary Arteries of Patients With Aortic Stenosis vs Insufficiency Undergoing a Ross Procedure.

BACKGROUND: Clinical studies have revealed a greater risk of pulmonary autograft dilation after the Ross procedure in patients with preoperative aortic insufficiency (AI). The present study examined whether the morphologic, biomechanical, and cellular properties of the pulmonary artery (PA) from patients with AI were phenotypically different compared with patients diagnosed with aortic stenosis (AS). METHODS: PA segments were harvested from patients undergoing the Ross procedure for AS (n = 16) and AI (n = 6). Preoperative aortic annulus was significantly larger (P < 0.05) in patients with AI (28.5 ± 1.8 mm) vs AS (22.8 ± 1.2 mm). Morphologic, biomechanical, and cellular phenotypes of the PA were analyzed. RESULTS: Collagen and elastin content in the media of the PA wall were similar in patients with AS and AI. Elastic modulus and energy loss of the PA were not significantly different between the groups. In the media of the PA, expression of a panel of vascular smooth muscle cell-specific proteins were similar in patients with AS and AI. In contrast, nonmuscle myosin IIB protein levels in the PA of AS patients were significantly higher compared with AI patients, and immunofluorescence identified staining in α-smooth muscle actin-positive vascular smooth muscle cells. CONCLUSIONS: Despite similar morphological and biomechanical properties, the disparate expression of nonmuscle myosin IIB protein distinguishes the PA of patients with AI from patients with AS. The biological role in vascular smooth muscle cells and the potential contribution of nonmuscle myosin IIB to pulmonary autograft dilation in a subset of AI patients after the Ross procedure remain to be determined.

p38α MAPK inhibition translates to cell cycle re-entry of neonatal rat ventricular cardiomyocytes and de novo nestin expression in response to thrombin and after apex resection.

The present study tested the hypothesis that p38α MAPK inhibition leads to cell cycle re-entry of neonatal ventricular cardiomyocytes (NNVMs) and de novo nestin expression in response to thrombin and after apex resection of the neonatal rat heart. Thrombin (1 U/ml) treatment of 1-day old NNVMs did not induce cell cycle re-entry or nestin expression. Acute exposure of NNVMs to thrombin increased p38α MAPK and HSP27 phosphorylation and p38α/ß MAPK inhibitor SB203580 abrogated HSP27 phosphorylation. Thrombin and SB203580 co-treatment of NNVMs led to bromodeoxyuridine incorporation and nestin expression. SB203580 (5 mg/kg) administration immediately after apex resection of 1-day old neonatal rat hearts and continued for two additional days shortened the fibrin clot length sealing the exposed left ventricular chamber. SB203580-treatment increased the density of troponin-T(+)-NNVMs that incorporated bromodeoxyuridine and expressed nuclear phosphohistone-3. Nestin(+)-NNVMs were selectively detected at the border of the fibrin clot and SB203580 potentiated the density that re-entered the cell cycle. These data suggest that the greater density of ventricular cardiomyocytes and nestin(+)-ventricular cardiomyocytes that re-entered the cell cycle after SB203580 treatment of the apex-resected neonatal rat heart during the acute phase of fibrin clot formation may be attributed in part to inhibition of thrombin-mediated p38α MAPK signalling.

Nestin expression is dynamically regulated in cardiomyocytes during embryogenesis.

The transcriptional factors implicated in the expression of the intermediate filament protein nestin in cardiomyocytes during embryogenesis remain undefined. In the heart of 9,5-10,5 day embryonic mice, nestin staining was detected in atrial and ventricular cardiomyocytes and a subpopulation co-expressed Tbx5. At later stages of development, nestin immunoreactivity in cardiomyocytes gradually diminished and was absent in the heart of 17,5 day embryonic mice. In the heart of wild type 11,5 day embryonic mice, 54 ± 7% of the trabeculae expressed nestin and the percentage was significantly increased in the hearts of Tbx5+/- and Gata4+/- embryos. The cell cycle protein Ki67 and transcriptional coactivator Yap-1 were still prevalent in the nucleus of nestin(+) -cardiomyocytes identified in the heart of Tbx5+/- and Gata4+/- embryonic mice. Phorbol 12,13-dibutyrate treatment of neonatal rat ventricular cardiomyocytes increased Yap-1 phosphorylation and co-administration of the p38 MAPK inhibitor SB203580 led to significant dephosphorylation. Antagonism of dephosphorylated Yap-1 signalling with verteporfin inhibited phorbol 12,13-dibutyrate/SB203580-mediated nestin expression and BrdU incorporation of neonatal cardiomyocytes. Nestin depletion with an AAV9 containing a shRNA directed against the intermediate filament protein significantly reduced the number of neonatal cardiomyocytes that re-entered the cell cycle. These findings demonstrate that Tbx5- and Gata4-dependent events negatively regulate nestin expression in cardiomyocytes during embryogenesis. By contrast, dephosphorylated Yap-1 acting via upregulation of the intermediate filament protein nestin plays a seminal role in the cell cycle re-entry of cardiomyocytes. Based on these data, an analogous role of Yap-1 may be prevalent in the heart of Tbx5+/- and Gata4+/- mice.

Nestin expression is upregulated in the fibrotic rat heart and is localized in collagen-expressing mesenchymal cells and interstitial CD31(+)- cells.

Renal and lung fibrosis was characterized by the accumulation of collagen-immunoreactive mesenchymal cells expressing the intermediate filament protein nestin. The present study tested the hypothesis that nestin expression was increased in the hypertrophied/fibrotic left ventricle of suprarenal abdominal aorta constricted adult male Sprague-Dawley rats and induced in ventricular fibroblasts by pro-fibrotic peptide growth factors. Nestin protein levels were upregulated in the pressure-overloaded left ventricle and expression positively correlated with the rise of mean arterial pressure. In sham and pressure-overloaded hearts, nestin immunoreactivity was detected in collagen type I(+)-and CD31(+)-cells identified in the interstitium and perivascular region whereas staining was absent in smooth muscle α-actin(+)-cells. A significantly greater number of collagen type I(+)-cells co-expressing nestin was identified in the left ventricle of pressure-overloaded rats. Moreover, an accumulation of nestin(+)-cells lacking collagen, CD31 and smooth muscle α-actin staining was selectively observed at the adventitial region of predominantly large calibre blood vessels in the hypertrophied/fibrotic left ventricle. Angiotensin II and TGF-ß1 stimulation of ventricular fibroblasts increased nestin protein levels via phosphatidylinositol 3-kinase- and protein kinase C/SMAD3-dependent pathways, respectively. CD31/eNOS(+)-rat cardiac microvascular endothelial cells synthesized/secreted collagen type I, expressed prolyl 4-hydroxylase and TGF-ß1 induced nestin expression. The selective accumulation of adventitial nestin(+)-cells highlighted a novel feature of large vessel remodelling in the pressure-overloaded heart and increased appearance of collagen type I/nestin(+)-cells may reflect an activated phenotype of ventricular fibroblasts. CD31/collagen/nestin(+)-interstitial cells could represent displaced endothelial cells displaying an unmasked mesenchymal phenotype, albeit contribution to the reactive fibrotic response of the pressure-overloaded heart remains unknown.

Nestin Expressed by Pre-Existing Cardiomyocytes Recapitulated in Part an Embryonic Phenotype; Suppressive Role of p38 MAPK.

Nestin(+) -cardiomyocytes were identified in the ischemically damaged human/rodent heart, albeit the cellular source, and signaling events implicated in the appearance of the intermediate filament protein remained undefined. Expression of the enhanced green fluorescent protein (EGFP) driven by the second intron of the nestin gene identified a subpopulation of EGFP/nestin(+) cells that differentiated to a vascular phenotype in the peri-infarct/infarct region of post-MI mice albeit the transgene was not detected in nestin(+) -cardiomyocytes. α-MHC-driven expression of the reporter mCherry was detected in troponin-T(+) - and nestin(+) -cardiomyocytes in the peri-infarct/infarct region of post-MI mice. However, the cell cycle re-entry of nestin/mCherry(+) -cardiomyocytes was not observed. Nestin staining was identified in a paucity of neonatal rat ventricular cardiomyocytes (NNVM). Exposure to phorbol 12,13-dibutyrate (PDBu) induced NNVM hypertrophy but did not promote nestin expression or Brdu incorporation. PDBu treatment of NNVMs phosphorylated p38 MAPK and HSP27 and HSP27 phosphorylation was abrogated by the p38 MAPK inhibitor SB203580. PDBu/SB203580 co-treatment significantly increased the percentage of NNVMs that expressed nestin and incorporated Brdu. In the heart of embryonic 10.5 day mice, nestin immunoreactivity was observed in cycling troponin-T(+) -cardiomyocytes. Nestin was also detected in embryonic rat ventricular cardiomyocytes and depletion of the intermediate filament protein attenuated cell cycle re-entry. Thus, nestin expressed by pre-existing cardiomyocytes following ischemic damage recapitulated in part an embryonic trait and may provide the requisite phenotype to initiate cell cycle re-entry. However, the overt activation of the p38 MAPK pathway post-MI may in part limit the appearance and inhibit the cell cycle re-entry of nestin(+) -cardiomyocytes. J. Cell. Physiol. 232: 1717-1727, 2017. © 2016 Wiley Periodicals, Inc.

Endothelial and Epithelial Cell Transition to a Mesenchymal Phenotype Was Delineated by Nestin Expression.

Endothelial and epithelial cell transition to a mesenchymal phenotype was identified as cellular paradigms implicated in the appearance of fibroblasts and development of reactive fibrosis in interstitial lung disease. The intermediate filament protein nestin was highly expressed in fibrotic tissue, detected in fibroblasts and participated in proliferation and migration. The present study tested the hypothesis that the transition of endothelial and epithelial cells to a mesenchymal phenotype was delineated by nestin expression. Three weeks following hypobaric hypoxia, adult male Sprague-Dawley rats characterized by alveolar and perivascular lung fibrosis were associated with increased nestin protein and mRNA levels and marked appearance of nestin/collagen type I((+))-fibroblasts. In the perivascular region of hypobaric hypoxic rats, displaced CD31((+))-endothelial cells were detected, exhibited a mesenchymal phenotype and co-expressed nestin. Likewise, epithelial cells in the lungs of hypobaric hypoxic rats transitioned to a mesenchymal phenotype distinguished by the co-expression of E-cadherin and collagen. Following the removal of FBS from primary passage rat alveolar epithelial cells, TGF-ß1 was detected in the media and a subpopulation acquired a mesenchymal phenotype characterized by E-cadherin downregulation and concomitant induction of collagen and nestin. Bone morphogenic protein-7 treatment of alveolar epithelial cells prevented E-cadherin downregulation, suppressed collagen induction but partially inhibited nestin expression. These data support the premise that the transition of endothelial and epithelial cells to a mesenchymal cell may have contributed in part to the appearance nestin/collagen type I((+))-fibroblasts and the reactive fibrotic response in the lungs of hypobaric hypoxic rats.

Nestin upregulation characterizes vascular remodeling secondary to hypertension in the rat.

Proliferation and hypertrophy of vascular smooth muscle cells represent hallmark features of vessel remodeling secondary to hypertension. The intermediate filament protein nestin was recently identified in vascular smooth muscle cells and in other cell types directly participated in proliferation. The present study tested the hypothesis that vessel remodeling secondary to hypertension was characterized by nestin upregulation in vascular smooth muscle cells. Two weeks after suprarenal abdominal aorta constriction of adult male Sprague-Dawley rats, elevated mean arterial pressure increased the media area and thickness of the carotid artery and aorta and concomitantly upregulated nestin protein levels. In the normal adult rat carotid artery, nestin immunoreactivity was observed in a subpopulation of vascular smooth muscle cells, and the density significantly increased following suprarenal abdominal aorta constriction. Filamentous nestin was detected in cultured rat carotid artery- and aorta-derived vascular smooth muscle cells and an analogous paradigm observed in human aorta-derived vascular smooth muscle cells. ANG II and EGF treatment of vascular smooth muscle cells stimulated DNA and protein synthesis and increased nestin protein levels. Lentiviral short-hairpin RNA-mediated nestin depletion of carotid artery-derived vascular smooth muscle cells inhibited peptide growth factor-stimulated DNA synthesis, whereas protein synthesis remained intact. These data have demonstrated that vessel remodeling secondary to hypertension was characterized in part by nestin upregulation in vascular smooth muscle cells. The selective role of nestin in peptide growth factor-stimulated DNA synthesis has revealed that the proliferative and hypertrophic responses of vascular smooth muscle cells were mediated by divergent signaling events.

Nestin is a marker of lung remodeling secondary to myocardial infarction and type I diabetes in the rat.

Upregulation of the intermediate filament protein nestin was identified in a subpopulation of fibroblasts during reactive and reparative fibrosis and directly contributed to the enhanced proliferative phenotype. The present study tested the hypothesis that nestin was expressed in lung fibroblasts and the pattern of expression represented a distinct marker of pulmonary remodeling secondary to myocardial infarction and type I diabetes. Nestin((+)) fibroblasts were detected in rat lungs and a subpopulation exhibited a myofibroblast phenotype delineated by the co-expression of smooth muscle α-actin. In the lungs of myocardial infarcted rats, interstitial collagen content and nestin mRNA/protein levels were significantly increased despite the absence of secondary pulmonary hypertension, whereas smooth muscle α-actin protein expression was unchanged. Exposure of rat pulmonary fibroblasts to pro-fibrotic stimuli angiotensin II and transforming growth factor-ß significantly increased nestin protein levels. In the lungs of type I diabetic rats, the absence of a reactive fibrotic response was associated with a significant downregulation of nestin mRNA/protein expression. Nestin was reported a target of miR-125b, albeit miR-125b levels were unchanged in pulmonary fibroblasts treated with pro-fibrotic stimuli. Nestin((+)) cells lacking smooth muscle α-actin/collagen staining were also identified in rodent lungs and a transgenic approach revealed that expression of the intermediate filament protein was driven by intron 2 of the nestin gene. The disparate regulation of nestin characterized a distinct pattern of pulmonary remodeling secondary to myocardial infarction and type I diabetes and upregulation of the intermediate filament protein in lung fibroblasts may have facilitated in part the reactive fibrotic response.

Nestin downregulation in rat vascular smooth muscle cells represents an early marker of vascular disease in experimental type I diabetes.

BACKGROUND: Nestin was reported to directly contribute to cell proliferation and the intermediate filament protein was detected in vascular smooth muscle cells. In experimental type I diabetes, nestin downregulation in the heart was identified as an incipient pathophysiological event. The following study tested the hypothesis that dysregulation of nestin expression in vascular smooth muscle cells represented an early event of vascular disease in experimental type I diabetes. METHODS/RESULTS: In the carotid artery and aorta of adult male Sprague-Dawley rats, a subpopulation of vascular smooth muscle cells co-expressed nestin and was actively involved in the cell cycle as reflected by the co-staining of nuclear phosphohistone-3. The infection of aortic vascular smooth muscle cells with a lentivirus containing a shRNAmir directed against nestin significantly reduced protein expression and concomitantly attenuated basal DNA synthesis. Two weeks following injection of adult male Sprague-Dawley rats with streptozotocin, the endothelial response of aortic rings to acetylcholine, vascular morphology and the total density of vascular smooth muscle cells in the vasculature of type I diabetic rats were similar to normal rats. By contrast, nestin protein levels and the density of nestin(+)/phosphohistone-3(+)-vascular smooth muscle cells were significantly reduced in type I diabetic rats. The in vivo observations were recapitulated in vitro as exposure of vascular smooth muscle cells to 30 mM D-glucose inhibited DNA synthesis and concomitantly reduced nestin protein expression. CONCLUSIONS: Hyperglycaemia-mediated nestin downregulation and the concomitant reduction of cycling vascular smooth muscle cells represent early markers of vascular disease in experimental type I diabetes.

The neurogenic response of cardiac resident nestin(+) cells was associated with GAP43 upregulation and abrogated in a setting of type I diabetes.

BACKGROUND: Cardiac nestin(+) cells exhibit properties of a neural progenitor/stem cell population characterized by the de novo synthesis of neurofilament-M in response to ischemic injury and 6-hydroxydopamine administration. The induction of growth associated protein 43 (GAP43) was identified as an early event of neurogenesis. The present study tested the hypothesis that the de novo synthesis of neurofilament-M by nestin(+) cells was preceded by the transient upregulation of GAP43 during the acute phase of reparative fibrosis in the infarcted male rat heart. Secondly, a seminal feature of diabetes is impaired wound healing secondary to an inadequate neurogenic response. In this regard, an additional series of experiments tested the hypothesis that the neurogenic response of cardiac nestin(+) cells was attenuated in a setting of type I diabetes. METHODS: The neurogenic response of cardiac nestin(+) cells was examined during the early phase of reparative fibrosis following permanent ligation of the left anterior descending coronary artery in the adult male rat heart. The experimental model of type I diabetes was created following a single injection of streptozotocin in adult male rats. The impact of a type I diabetic environment on the neurogenic response of cardiac nestin(+) cells was examined during myocardial infarction and following the administration of 6-hydroxydopamine. RESULTS: During the early phase of scar formation/healing, the density of GAP43/nestin(+) fibres innervating the peri-infarct/infarct region was significantly increased, whereas neurofilament-M/nestin(+) fibres were absent. With ongoing scar formation/healing, a temporal decrease of GAP43/nestin(+) fibre density and a concomitant increase in the density of innervating neurofilament-M/nestin(+) fibres were observed. The neurogenic response of cardiac nestin(+) cells during scar formation/healing was inhibited following the superimposition of type I diabetes. The de novo synthesis of neurofilament-M by nestin(+) cells after 6-hydroxydopamine administration was likewise attenuated in the heart of type I diabetic rats whereas the density of GAP43/nestin(+) fibres remained elevated. CONCLUSION: The transient upregulation of GAP43 apparently represents a transition event during the acquisition of a neuronal-like phenotype and a type I diabetic environment attenuated the neurogenic response of cardiac nestin(+) cells to ischemia and 6-hydroxydopamine.