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Biomedical subjects

K R Stenmark

Publications and source records attributed to K R Stenmark.

At least 19 recordsLinked to original sources
PubMed1999

Relationship between perlecan and tropoelastin gene expression and cell replication in the developing rat pulmonary vasculature.

Smooth-muscle-cell (SMC) replication and extracellular matrix protein expression are two vital and interrelated processes necessary for normal development of the vasculature. To understand better the nature of this relationship in the developing rat lung, we investigated the relationship between SMC proliferation and the expression of perlecan, a basement membrane (BM) heparan sulfate proteoglycan implicated in the control of SMC growth and differentiation, and tropoelastin (TE), a structural matrix protein not known to influence directly the replicative state of SMCs. Using bromodeoxyuridine (BrdU) incorporation to assess DNA synthesis, we first established the time course of SMC proliferation in the hilar pulmonary artery (PA) from embryonic to adult life. We found a labeling index of > 80% during the embryonic period (embryonic Day 13 [e13] to fetal Day 18 [f18]), a dramatic decline to approximately 40% during the fetal period of development, and a steady decrease in proliferation rates following birth such that, by 30 d of age, a labeling index of < 2% was noted. Using in situ hybridization, we found that although peak expression of both perlecan and TE messenger RNA (mRNA) occurred in the fetal and early postnatal periods following the major decrease in cell replication, TE mRNA expression was clearly observed in the PA as early as embryonic Day 14, whereas perlecan transcripts were virtually undetectable until fetal Day 19. Therefore, to evaluate further the relationship between cell replication and perlecan and/or TE gene expression, we used a combined in situ hybridization/BrdU immunohistochemistry technique and demonstrated that, on an individual cell basis, perlecan message was predominantly expressed by nonreplicating (BrdU-negative) PA, whereas TE mRNA was equally expressed in replicating and nonreplicating PA SMCs. Interestingly, a very similar pattern of replication and relationship to perlecan and TE mRNA expression was noted in airway SMCs and epithelial cells. Thus, in the lung as a whole, maximal expression of both the BM protein perlecan and the interstitial matrix protein TE occurs coordinately and follows the period of maximal SMC proliferation. However, in individual SMCs, perlecan mRNA expression varies inversely with DNA synthesis, whereas TE mRNA expression appears independent of the proliferative state of the cell.

Animals↗
PubMed1998

Lung endothelial cell proliferation in normal and pulmonary hypertensive neonatal calves.

Tremendous changes in pressure and flow occur in the pulmonary and systemic circulations after birth, and these hemodynamic changes should markedly affect endothelial cell replication. However, in vivo endothelial replication rates in the neonatal period have not been reported. To label replicating endothelial cells, we administered the thymidine analog bromodeoxyuridine to calves approximately 1, 4, 7, 10, and 14 days old before they were killed. Because we expected the ratio of replicating to nonreplicating cells to vary with vascular segment, we examined the main pulmonary artery, a large elastic artery, three sizes of intrapulmonary arteries, the aorta, and the carotid artery. In normoxia for arteries < 1,500 micron, approximately 27% of the endothelial cells were labeled on day 1 but only approximately 2% on day 14. In the main pulmonary artery, only approximately 4% of the endothelial cells were labeled on day 1 and approximately 2% on day 14. In contrast, in the aorta, approximately 12% of the endothelial cells were labeled on day 1 and approximately 2% on day 14. In chronically hypoxic animals, only approximately 14% of the endothelial cells were labeled on day 1 in small lung arteries and approximately 8% were still labeled on day 14. We conclude that the postnatal circulatory adaptation to extrauterine life includes significant changes in endothelial cell proliferation that vary dramatically with time and vascular location and that these changes are altered in chronic hypoxia.

Animals↗
PubMed1997

Right ventricular angiotensin converting enzyme activity and expression is increased during hypoxic pulmonary hypertension.

OBJECTIVE: To determine whether local cardiac angiotensin converting enzyme (ACE) expression is upregulated during the development of hypoxia-induced right ventricular hypertrophy. METHODS: ACE activity was measured in membrane preparations from the right ventricle and left ventricle plus septum in normoxic rats and animals exposed to chronic hypoxia for 8 and 14 days. Local cardiac ACE expression was studied by immunohistochemistry using a monoclonal antibody to ACE (9B9). RESULTS: In the normal rat heart, ACE expression was confined to vascular endothelium, the valvular endocardium, and localized regions of parietal endocardium. We found that the development of pulmonary hypertension and right ventricular hypertrophy were associated with 2.6- and 3.4-fold increases in membrane-bound right ventricular ACE activity by 8 and 14 days of hypoxia, respectively. Right ventricular ACE activity was positively correlated with the degree of right ventricular hypertrophy (r = 0.83, P < 0.001). In contrast, left ventricular plus septal ACE activity was significantly reduced by approximately 40 and 60% by 8 and 14 days of hypoxia, respectively, compared to controls. In the right ventricle of chronically hypoxic rats, immunohistochemistry demonstrated increased ACE expression in areas of myocardial fibrosis. Interestingly, increased ACE expression was noted in the right ventricular epicardium in chronically hypoxic rats. In the free wall of the left ventricle there was a significant reduction in the number of myocardial capillaries which expressed ACE in chronically hypoxic rats. CONCLUSION: Chronic hypoxia has a differential effect on left and right ventricular ACE activity and that the sites of altered ACE expression are highly localized. We speculate that locally increased right ventricular ACE activity and expression may play a role in the pathogenesis of right ventricular hypertrophy secondary to hypoxic pulmonary hypertension.

Animals↗
PubMed1997

Cellular and molecular mechanisms of pulmonary vascular remodeling.

In many organs and tissues, the cellular response to injury is associated with a reiteration of specific developmental processes. Studies have shown that, in response to injury, vascular wall cells in adult organisms express genes or gene products characteristic of earlier developmental states. Other genes, expressed preferentially in adult cells in vivo, are down-regulated following injurious stimuli. Complicating matters, however, are recent observations demonstrating that the vascular wall is comprised of phenotypically heterogeneous subpopulations of endothelial cells, smooth muscle cells, and fibroblasts. It is unclear how specific subsets of cells respond to injury and thus contribute to the vascular remodeling that characterizes chronic pulmonary hypertension. This review discusses vascular development in the lung and the cellular responses occurring in pulmonary hypertension; special attention is given to heterogeneity of responses within cell populations and reiteration of developmental processes.

Animals↗
PubMed1997

Pulmonary artery smooth muscle cells from chronically hypoxic neonatal calves retain fetal-like and acquire new growth properties.

Growth properties retained and acquired by immature pulmonary artery (PA) smooth muscle cells (SMC) in vivo after chronic exposure to hypoxia and the mechanisms that regulate hypoxia-induced change in proliferative phenotype are not known. We tested the hypothesis that PA SMC from neonatal calves exposed to hypoxia after birth would both retain fetal-like and acquire new growth characteristics and that these changes would be at least partially dependent on protein kinase C (PKC), a key proproliferative signal transduction pathway. Like fetal cells, PA SMC from hypoxic calves grew faster in the presence and absence of serum and were more responsive to insulin-like growth factor I and platelet-derived growth factor-BB than control neonatal and adult cells. PA SMC from hypoxic calves also acquired other growth properties (i.e., including increased hypoxic growth after PKC activation) that were new compared with those observed for fetal cells. The proliferative response to hypoxia was first detectable in the neonatal period and was further increased in cells from hypoxic calves. SMC from fetuses and hypoxic calves were more susceptible to the growth-inhibiting effects of PKC antagonists (dihydrosphingosine and calphostin C) than control neonatal and adult cells. To test if the Ca(2+)-dependent isozymes of PKC were uniquely important in the developmental and acquired growth changes observed, the antagonistic effect of the specific, but isozyme nonselective, PKC inhibitor Ro-81-8220 was then compared with GF-109203X, a structural analog with relative specificity for the Ca(2+)-dependent isozymes of PKC (alpha and beta in PA SMC). The faster growing PA SMC from bovine fetuses and hypoxia-exposed calves again demonstrated greater growth inhibition in response to both inhibitors. GF-109203X was equipotent to Ro-31-8220, and its antiproliferative effects were shown to not be due to an increase in apoptosis. Phorbol ester-induced PKC downregulation, another inhibitor strategy that selectively depletes bovine PA SMC of PKC-alpha, but not -beta, mimicked the antiproliferative effects of GF-109203X. Whole cellular PKC catalytic activity paralleled the pattern of peptide-induced growth and susceptibility to PKC inhibition. These results suggest that PA SMC from hypoxia-exposed neonatal calves retain enhanced fetal-like proliferative capacity and acquire new growth properties that are at least partially dependent on the Ca(2+)-regulated isozymes of PKC and in particular PKC-alpha.

Aging↗
PubMed1997

Selected isozymes of PKC contribute to augmented growth of fetal and neonatal bovine PA adventitial fibroblasts.

We sought to determine which isozymes of protein kinase C (PKC) contribute to the increased proliferation of immature bovine pulmonary artery (PA) adventitial fibroblasts. Seven were identified in lysates of neonatal PA fibroblasts by Western blot: three Ca2+ dependent (alpha, beta I, and beta II) and four Ca2+ independent (delta, epsilon, zeta, and mu). Four isozymes (gamma, eta, theta, and iota) were not detected in fibroblasts isolated at any developmental stage. Of the seven detected isozymes, only PKC-alpha and -beta II protein levels were higher in fetal and neonatal cells compared with adult fibroblasts. Their role in the enhanced growth of immature fibroblasts was then evaluated. The isozyme nonselective PKC inhibitor Ro-31-8220 was first compared with GF-109203X, a structural analog of Ro-31-8220 with relative specificity for the Ca(2+)-dependent isozymes of PKC. GF-109203X selectively inhibited the growth of immature cells and was nearly as potent as Ro-31-8220. Go-6976, a more specific inhibitor of the Ca(2+)-dependent isozymes, mimicked the antiproliferative effect of GF-109203X. PKC downregulation with 1 microM phorbol 12-myristate 13-acetate had the same selective antiproliferative effect on immature fibroblasts as GF-109203X and Go-6976. The protein levels of PKC-alpha and -beta II, but not of PKC-beta I, were completely degraded in response to phorbol 12-myristate 13-acetate pretreatment. These results suggest that PKC-alpha and -beta II are important in the augmented growth of immature bovine PA adventitial fibroblasts.

Aging↗
PubMed1997

Smooth muscle cell heterogeneity in pulmonary and systemic vessels. Importance in vascular disease.

Experimental evidence is rapidly accumulating which demonstrates that the arterial media in both pulmonary and systemic vessels is not composed of a phenotypically homogeneous population of smooth muscle cells (SMCs) but rather of heterogeneous subpopulations of cells with unique developmental lineages. In vivo and in vitro observations strongly suggest that marked differences in the phenotype, growth, and matrix-producing capabilities of phenotypically distinct SMC subpopulations exist and that these differences are intrinsic to the cell type. These data also suggest that differential proliferative and matrix-producing capabilities of distinct SMC subpopulations govern, at least in part, the pattern of abnormal cell proliferation and matrix protein synthesis observed in the pathogenesis of vascular disease. Within the pulmonary circulation, the observation that the isolated medial SMC subpopulations exhibit differential proliferative responses to hypoxic exposure is important, since this in vitro cell-model system can now be used to better understand the mechanisms that regulate increased responsiveness of specific medial cell subpopulations to low oxygen concentrations. Our data also support the idea that protein kinase C is likely to be one important determinant of differential cell growth responses to hypoxia. The data also suggest differential involvement of specific arterial SMC subpopulations in the elastogenic responses of the vessel wall to injury. We believe that a better understanding of the mechanisms contributing to the unique behavior of specific arterial cell subpopulations will provide important future directions for therapies aimed at preventing abnormal cell replication and matrix protein synthesis in vascular disease.

Actins↗
PubMed1997

Smooth muscle cells isolated from discrete compartments of the mature vascular media exhibit unique phenotypes and distinct growth capabilities.

Heterogeneity of smooth muscle cell (SMC) phenotype and function is rapidly emerging as an important concept. We have recently described that phenotypically distinct SMC subpopulations in bovine pulmonary arteries exhibit unique proliferative and matrix-producing responses to hypoxic pulmonary hypertension. To provide better understanding of the molecular mechanisms contributing to this phenomenon, experimental studies will require a reliable in vitro model. The purpose of the present study was first to determine if distinct SMC subpopulations, similar to those observed in vivo, could be selectively isolated from the mature arterial media, and then to evaluate whether select SMC subpopulations would exhibit heightened responses to growth-promoting stimuli and hypoxia. We were able to reproducibly isolate at least four phenotypically unique cell subpopulations from the inner, middle, and outer compartments of the arterial media. Differences in cell phenotype were demonstrated by morphological appearance and differential expression of muscle-specific proteins. The isolated cell subpopulations exhibited markedly different growth capabilities. Two SMC subpopulations grew slowly in 10% serum and were quiescent in plasma-based medium. The other two cell subpopulations, exhibiting nonmuscle characteristics, grew rapidly in 10% serum and proliferated in plasma-based medium and in response to hypoxia. Certain colonies of the nonmuscle-like cell subpopulations were found to grow autonomously under serum-deprived conditions and to secrete mitogenic factors. Our data, demonstrating that phenotypically distinct cells with enhanced growth potential exist within the normal arterial media, support the idea that these unique cells could contribute selectively to the pathogenesis of vascular disease.

Actins↗
PubMed1997

Hypoxia increases bromodeoxyuridine labeling indices in bovine neonatal pulmonary arteries.

Thickening of peripheral pulmonary arteries (PA) in the pulmonary hypertensive neonate has been well described morphologically, but less is known regarding the role of cell proliferation in either the normal or hypertensive neonatal PA. Thus we studied DNA synthetic indices in the tunica media and tunica adventitia of four different sizes/generations of PA in normoxic calves (n = 15) and calves exposed to hypobaric hypoxia (n = 15) during the first 14 days of life. DNA synthetic indices were determined by incorporation of the thymidine analogue bromodeoxyuridine (BrdU). Hemodynamic studies confirmed a steady decline in PA pressure in normal neonatal calves during the first 2 wk of life and progressive pulmonary hypertension in the hypoxic group. Lungs were perfusion-fixed and pulmonary arteries were evaluated for BrdU incorporation by immunohistochemistry. DNA synthetic indices (BrdU-labeled cells/1,000 cells) in the tunica media from normoxic calves were highest between 4 and 7 days postpartum and decreased to their lowest levels by day 14. The highest indices were observed in smaller generations of PA in the normoxic newborn. Adventitial cells exhibited the same general pattern of BrdU incorporation except that the postpartum peak occurred earlier, at 1 to 4 days. Exposure to hypoxia significantly increased (P = 0.001) DNA synthetic indices in both the tunica media and adventitia. The highest DNA synthetic indices were observed in smaller-generation vessels. These findings indicate that the fraction of cells traversing the S phase (i.e., actively replicating in the cell cycle) in the normal neonatal pulmonary vasculature during transition are initially high compared to reported rates in hilar PA from adult rats, but then decrease by 14 days after birth. Further, exposure to hypoxia during transition dramatically increases and prolongs pulmonary vascular cell proliferation. We conclude that structural remodeling in the hypertensive neonatal PA is due partly to increased cell proliferation in the tunica media and adventitia.

Animals↗
PubMed1997

High-dose epinephrine is not superior to standard-dose epinephrine in pediatric in-hospital cardiopulmonary arrest.

OBJECTIVE: To compare the efficacy of high-dose epinephrine (HDE) with that of standard-dose epinephrine (SDE) for resuscitation from in-hospital pediatric cardiopulmonary arrest (CPA). DESIGN: Fifty-four-month retrospective study of all pediatric patients who had a CPA while hospitalized at a tertiary care children's hospital. Standard pediatric advanced life support techniques were used for all patients. Patients received HDE or SDE in accordance with physician orders and standard protocols at the time of CPA. Primary outcome measures were the return of spontaneous circulation (ROSC), the duration of survival after resuscitation, survival to hospital discharge, and Pediatric Overall Performance Category scores at the time of discharge. RESULTS: During the study period, 51 patients met entry criteria and had a total of 58 CPAs. Twenty-one patients received HDE during resuscitation from 24 arrests, at a dose of 0.12 +/- 0.05 mg/kg (mean +/- SD); 30 patients received SDE during resuscitation from 34 arrests, at a dose of 0.01 +/- 0.01 mg/kg (mean +/- SD). The HDE and SDE groups were not significantly different in terms of gender, initial cardiac rhythm, location of CPA, primary diagnoses at the time of CPA, initial pH, or additional resuscitation medications received; the SDE group had a significantly higher mean age, although the median ages were not different. Fourteen of 24 resuscitations using HDE resulted in ROSC (58%) with a mean time to ROSC of 19 minutes; 7 (29%) of 24 led to survival for 24 hours, and 6 (26%) of 23 patients survived to hospital discharge, all with moderate to severe neurologic and functional impairment. Twenty-four of 34 resuscitations using SDE resulted in ROSC (71%) with a mean time to ROSC of 12 minutes; 17 (50%) of 34 led to survival for 24 hours; and 7 (23%) of 30 patients survived to hospital discharge, 4 with mild to moderate neurologic impairment. No significant differences in rates of ROSC, survival rates, or Pediatric Overall Performance Category scores of survivors were found between the two groups. The mean time to ROSC was significantly longer in the HDE group. CONCLUSIONS: In this study, the use of HDE did not improve the rates of ROSC, short-term survival, or long-term survival after pediatric in-hospital CPA, nor did it improve overall outcome scores. Given the conflicting evidence surrounding possible detrimental effects of HDE use, a large, blinded, prospective trial of HDE use in this setting is necessary to clarify the appropriate role for HDE in pediatric resuscitation.

Child, Preschool↗
PubMed1997

Heterogeneity in the proliferative response of bovine pulmonary artery smooth muscle cells to mitogens and hypoxia: importance of protein kinase C.

Pulmonary artery (PA) smooth muscle cell (SMC) proliferation is an important contributor to the vascular remodeling that occurs in chronic hypoxic pulmonary hypertension. The earliest SMC proliferative changes in response to hypoxia occur in the outer media. We tested the hypothesis that the pattern of hypoxia-induced PA SMC proliferation observed in vivo is determined at least in part by intrinsic differences in proliferative response of SMC isolated from different medial layers to relevant peptide mitogens and hypoxia. Adult bovine PA SMCs were isolated at the same proximal site from the middle (layer 2) and outer (layer 3) media. In response to maximal serum stimulation, PA SMCs from the outer media grew faster than cells from the middle media. The outer medial cells also had increased responsiveness to multiple peptide mitogens (IGF-I, PDGF-BB, bFGF, and EGF). Because protein kinase C (PKC), a key pro-proliferative signal transduction pathway, has been shown to play an important role in this type of global increase in growth, responsiveness to a direct cell-permeable activator of PKC (PMA, phorbol 12-myristate 13-acetate) was then measured. PA SMCs from the outer media had greater DNA synthesis in response to selective PKC activation than middle medial cells. Since activation of this kinase is a requisite step for PA SMCs to proliferate in response to hypoxia, the hypoxic growth potential of cells from the middle and outer media was then compared. SMCs from the outer media had an augmented proliferative response to hypoxia compared with those from the middle media. These data suggested an important role for PKC in the enhanced growth of PA SMCs from the outer media. Therefore, whole cellular activity, expression, and hypoxia-induced activation of PKC were measured in both subpopulations of PA SMCs. Outer medial cells had greater total cellular activity, expression, and hypoxia-induced activation of PKC (and the alpha isozyme in particular) than cells isolated from the middle media. These findings support the concept that heterogeneity in growth capacity of PA SMCs exists within the bovine PA media, that these intrinsic differences in growth govern, at least in part, the pattern of abnormal SMC proliferation observed in vivo, and that the PKC pathway (and PKC-alpha in particular) is likely an important determinant of the subpopulation-specific differences found.

Animals↗
PubMed1996

Developmental regulation of angiotensin converting enzyme and angiotensin type 1 receptor in the rat pulmonary circulation.

Factors that influence the development of the normal pulmonary vasculature are poorly understood. Since increased local production of angiotensin II (AII) by angiotensin converting enzyme (ACE) has been implicated in the medial hypertrophy of systemic and pulmonary hypertension, we questioned whether ACE and angiotensin receptor expression may influence the muscularization of the normal pulmonary vasculature during development. The approach employed measurement of lung ACE activity, assessment of local ACE expression by immunohistochemistry, and angiotensin type 1 receptor (AT1) expression by in situ hybridization in rat lungs ranging from 15 days of intrauterine life (term = 21 d) to adulthood. The temporal and spatial pattern of ACE expression was compared with that of the endothelial marker, von Willebrand factor (vWF), and the smooth muscle cell markers, alpha smooth muscle actin and smooth muscle myosin. ACE activity was first detected in lung homogenates on day 17 of gestation (1 +/- 0.2 mU/mg) and increased progressively to term (27.7 +/- 3.2 mU/mg). However, the greatest increase in lung ACE activity to adult levels (379 +/- 25.2 mU/mg) occurred between 2 and 4 wk of postnatal life. Immunohistochemistry demonstrated vWF expression by vascular endothelium throughout the lung as early as day 15 of gestation. In contrast, ACE expression was observed in the endothelium of only hilar pulmonary arteries on day 15 of gestation, and thereafter was noted to be expressed in endothelial cells of progressively more distal arteries, such that by term, endothelial cells of all muscularized arteries expressed ACE. Alveolar capillary ACE expression was not detected until day 20 of gestation, and increased dramatically after birth. Smooth muscle actin expression in lung arteries closely paralleled the expression of endothelial ACE. AT1 receptor mRNA was first expressed in the peripheral lung on day 17 of gestation by non-epithelial undifferentiated mesenchyme. In contrast, AT1 mRNA signal was much reduced in differentiated smooth muscle. We speculate that ACE expression in the fetal lung circulation may influence the muscularization of fetal pulmonary arteries by the interaction of locally produced angiotensin II with the AT1 receptor.

Animals↗
PubMed1996

Expression and localization of tropoelastin mRNA in the developing bovine pulmonary artery is dependent on vascular cell phenotype.

During vascular development, the expression of tropoelastin (TE) messenger ribonucleic acid (mRNA) has been shown to be time dependent and to form complex patterns along the longitudinal and radial arterial axes. The factors contributing to these patterns of TE expression are not known, but it has been suggested that they reflect phenotypic changes in developing smooth muscle cells (SMC). In order to examine a possible correlation between the developmental state of the SMC and TE expression during lung vascular development, we localized and assessed relative TE mRNA expression in the developing bovine main pulmonary artery (PA), and correlated the observed patterns of TE expression to changes in SMC phenotype as determined by the expression of various developmentally related SMC proteins. Further, because TE expression can be modulated by physical forces such as pressure, fetal PA TE expression was evaluated with regard to changes in fetal arterial pressure. We found that expression of TE mRNA exhibited a biphasic pattern during fetal development. In early gestation, expression was noted throughout the entire PA wall; at midgestation, expression was markedly decreased in the outer wall but maintained in the inner vascular media; at late gestation, reexpression was observed throughout the entire PA wall, albeit in a heterogeneous pattern. Immunohistochemical studies showed that the decrease in SMC TE expression during midgestation coincided with the acquisition of SMC-specific proteins such as smooth muscle myosin heavy chains and desmin. The reexpression of TE late in gestation occurred in these "differentiated" SMC and was temporally associated with a large increase in arterial pressure shown to occur in late gestation. In addition, we identified an SMC population defined by its immunoreactivity to the muscle-specific cytoskeletal protein meta-vinculin that did not express TE mRNA either during fetal PA development or postnatally when PA hypertension was induced. We conclude that both the developmental state of the SMC and hemodynamic forces correlate with the pattern of PA TE mRNA expression during pulmonary vascular development. Further, a subpopulation of SMC defined by meta-vinculin expression exists in the fetal and neonatal bovine vascular wall and does not express detectable levels of TE mRNA regardless of vascular pressure.

Animals↗