Mark A. Perrella

Tumor necrosis factor downregulates an endothelial nitric oxide synthase mRNA by shortening its half-life.

Nitric oxide (NO), which accounts for the biological properties of endothelium-derived relaxing factor, is generated by NO synthase (NOS). The vascular endothelium contains two types of NOS: one is constitutively expressed (cNOS), and the other is inducible. Endothelium-mediated vasorelaxation is impaired in atherosclerotic vessels. To determine whether tumor necrosis factor (TNF)-alpha, which is commonly found in atherosclerotic lesions, has an effect on NOS message, we measured cNOS mRNA levels in TNF-treated human umbilical vein endothelial cells (HUVECs) by RNA blot analysis with a cNOS cDNA probe. TNF-alpha markedly reduced cNOS mRNA levels in HUVECs in a dose- and time-dependent manner. In response to 3 ng/mL TNF-alpha, cNOS mRNA levels began to decrease at 4 hours and diminished to only 5% of control levels at 24 hours. As little as 0.1 ng/mL TNF-alpha reduced cNOS mRNA levels by 50%. This reduction in cNOS message in response to TNF-alpha depended on protein synthesis as it was blocked by cycloheximide. In nuclear runoff experiments, TNF-alpha did not change the rate of cNOS gene transcription. cNOS mRNA is very stable under basal conditions, with a half-life of 48 hours; however, treatment with TNF-alpha shortened this half-life to 3 hours. TNF-alpha thus appears to decrease cNOS mRNA levels by increasing the rate of mRNA degradation. TNF-induced reductions in cNOS mRNA levels may have an important effect on impaired endothelium-mediated vasorelaxation in atherosclerosis.

Natriuretic peptide system in human heart failure.

BACKGROUND Atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP) are a family of structurally related peptides that participate in the integrated control of renal and cardiovascular function. Previous studies suggest a functional role for these hormonal peptides in cardiorenal regulation in congestive heart failure (CHF). METHODS AND RESULTS The present studies were performed in normal subjects (n = 6) and in patients with mild (New York Heart Association [NYHA] class I to II, n = 20) and severe (NYHA class III to IV, n = 20) CHF by use of radioimmunoassay and immunohistochemical staining (IHCS). Plasma ANP was significantly increased in both mild and severe CHF compared with normal subjects. In contrast, plasma BNP was only moderately increased in the severe CHF group, and plasma CNP concentration was unchanged in CHF compared with normal subjects. Atrial tissue concentrations of the natriuretic peptides did not parallel circulating concentrations. ANP predominated in normal atrial tissue, but BNP predominated in CHF. In ventricular tissue, IHCS staining was present for all three peptides in normal ventricular myocardium and was markedly enhanced in CHF. CONCLUSIONS These studies support a differential regulation of ANP, BNP, and CNP circulating concentrations and tissue activity in human CHF.

Hypoxia induces severe right ventricular dilatation and infarction in heme oxygenase-1 null mice

Heme oxygenase (HO) catalyzes the oxidation of heme to generate carbon monoxide (CO) and bilirubin. CO increases cellular levels of cGMP, which regulates vascular tone and smooth muscle development. Bilirubin is a potent antioxidant. Hypoxia increases expression of the inducible HO isoform (HO-1) but not the constitutive isoform (HO-2). To determine whether HO-1 affects cellular adaptation to chronic hypoxia in vivo, we generated HO-1 null (HO-1(-/-)) mice and subjected them to hypoxia (10% oxygen) for five to seven weeks. Hypoxia caused similar increases in right ventricular systolic pressure in wild-type and HO-1(-/-) mice. Although ventricular weight increased in wild-type mice, the increase was greater in HO-1(-/-) mice. Similarly, the right ventricles were more dilated in HO-1(-/-) mice. After seven weeks of hypoxia, only HO-1(-/-) mice developed right ventricular infarcts with organized mural thrombi. No left ventricular infarcts were observed. Lipid peroxidation and oxidative damage occurred in right ventricular cardiomyocytes in HO-1(-/-), but not wild-type, mice. We also detected apoptotic cardiomyocytes surrounding areas of infarcted myocardium by terminal deoxynucleotide transferase-mediated dUTP nick end-labeling (TUNEL) assays. Our data suggest that in the absence of HO-1, cardiomyocytes have a maladaptive response to hypoxia and subsequent pulmonary hypertension. J.Clin. Invest. 103:R23-R29 (1999).

Evidence for Human Lung Stem Cells

BACKGROUND Although progenitor cells have been described in distinct anatomical regions of the lung, description of resident stem cells has remained elusive. METHODS Surgical lung-tissue specimens were studied in situ to identify and characterize human lung stem cells. We defined their phenotype and functional properties in vitro and in vivo. RESULTS Human lungs contain undifferentiated human lung stem cells nested in niches in the distal airways. These cells are self-renewing, clonogenic, and multipotent in vitro. After injection into damaged mouse lung in vivo, human lung stem cells form human bronchioles, alveoli, and pulmonary vessels integrated structurally and functionally with the damaged organ. The formation of a chimeric lung was confirmed by detection of human transcripts for epithelial and vascular genes. In addition, the self-renewal and long-term proliferation of human lung stem cells was shown in serial-transplantation assays. CONCLUSIONS Human lungs contain identifiable stem cells. In animal models, these cells participate in tissue homeostasis and regeneration. They have the undemonstrated potential to promote tissue restoration in patients with lung disease. (Funded by the National Institutes of Health.).

Targeted expression of heme oxygenase-1 prevents the pulmonary inflammatory and vascular responses to hypoxia

Chronic hypoxia causes pulmonary hypertension with smooth muscle cell proliferation and matrix deposition in the wall of the pulmonary arterioles. We demonstrate here that hypoxia also induces a pronounced inflammation in the lung before the structural changes of the vessel wall. The proinflammatory action of hypoxia is mediated by the induction of distinct cytokines and chemokines and is independent of tumor necrosis factor-α signaling. We have previously proposed a crucial role for heme oxygenase-1 (HO-1) in protecting cardiomyocytes from hypoxic stress, and potent anti-inflammatory properties of HO-1 have been reported in models of tissue injury. We thus established transgenic mice that constitutively express HO-1 in the lung and exposed them to chronic hypoxia. HO-1 transgenic mice were protected from the development of both pulmonary inflammation as well as hypertension and vessel wall hypertrophy induced by hypoxia. Significantly, the hypoxic induction of proinflammatory cytokines and chemokines was suppressed in HO-1 transgenic mice. Our findings suggest an important protective function of enzymatic products of HO-1 activity as inhibitors of hypoxia-induced vasoconstrictive and proinflammatory pathways.

Enhanced Inhibition of Neointimal Hyperplasia by Genetically Engineered Endothelial Progenitor Cells

Background—Circulating endothelial progenitor cells (EPCs) have been reported previously. In this study, we examined the hypothesis that overexpression of vasculoprotective gene endothelial nitric oxide synthase (eNOS) and heme oxygenase-1 (HO-1) in EPCs enhances their ability to inhibit neointimal hyperplasia. Methods and Results—EPCs were isolated from rabbit peripheral blood, expanded in culture, and transduced with pseudotyped retroviral vectors expressing human eNOS (eNOS-EPCs), HO-1 (HO-1-EPCs), or green fluorescent protein (GFP-EPCs). Transduction efficiency of EPCs ex vivo was >90%. Four groups of rabbits (n=5 to 6 per group) were subjected to balloon angioplasty of the common carotid artery. Immediately after injury, ≈5×106 autologous eNOS-EPCs or HO-1-EPCs were transplanted into the injured vessel. Control animals received an equivalent number of GFP-EPCs or Ringer’s saline. Two weeks after transplantation, eNOS and HO-1 transgene transcripts and proteins were detected in the transduced rabbit vessels. Endothelialization was enhanced in the EPC-transplanted vessels independently of gene transfer. Neointimal thickening was significantly reduced in the GFP-EPC–treated vessels relative to the saline control. Neointima size was further reduced in vessels treated with eNOS-EPCs. Surprisingly, no additional reduction was seen in vessels treated with HO-1-EPCs relative to GFP-EPCs. Thrombosis occurred in ≈50% of the saline-treated vessels but was virtually absent in all EPC-transplanted vessels. Conclusions—We conclude that transplantation of autologous EPCs overexpressing eNOS in injured vessels enhances the vasculoprotective properties of the reconstituted endothelium, leading to inhibition of neointimal hyperplasia. This cell-based gene therapy strategy may be useful in treatment of vascular disease.

Increased endothelin in experimental heart failure.

Recent studies demonstrate that endothelin, a potent endogenous vasoconstrictor peptide, circulates in plasma of normal animals and humans. However, the role of this peptide in pathophysiological states remains unclear. The present study was designed to test the hypothesis that circulating endothelin concentrations are increased in experimental congestive heart failure (CHF), a pathophysiological state characterized by activation of vasoconstrictor mechanisms. In anesthetized dogs with CHF produced by 8 days of rapid ventricular pacing (n = 28), circulating plasma endothelin was increased compared with values for normal controls (n = 28; 20.4 +/- 1.4 versus 9.7 +/- 0.9 pg/ml, respectively; p less than 0.0001). A plasma endothelin level of more than 14.0 was a sensitive and specific indicator of significant CHF. Moreover, within the group with experimental CHF, right atrial pressure and pulmonary capillary wedge pressure correlated independently with circulating endothelin levels. Based on recent studies demonstrating the physiological actions of twofold increases in circulating endothelin, as observed in the present study, a possible role for endothelin in the pathophysiology of CHF is advanced.

Absence of heme oxygenase-1 exacerbates atherosclerotic lesion formation and vascular remodeling

To examine the role of heme oxygenase (HO)‐1 in the pathophysiology of vascular diseases, we generated mice deficient in both HO‐1 and apolipoprotein E (HO‐1−/−apoE−/−). Despite similar total plasma cholesterol levels in response to hypercholesterolemia, HO‐1−/−apoE−/− mice, in comparison with HO‐1+/+apoE−/− mice, had an accelerated and more advanced atherosclerotic lesion formation. In addition to greater lipid accumulation, these advanced lesions from HO‐1−/−apoE−/− mice contained macrophages and smooth muscle α‐actin‐positive cells. We further tested the role of HO‐1 on neointimal formation in a mouse model of vein graft stenosis. Autologous vein grafts in HO‐1−/− mice showed robust neointima consisting of α‐actin‐positive vascular smooth muscle cells (VSMC) 10 days after surgery in comparison to the smaller neointima formed in autologous vein grafts in HO‐1+/+ mice. However, at 14 days after surgery, the neointima from composite vessels of HO‐1−/− mice was composed mainly of acellular material, indicative of substantial VSMC death. VSMC isolated from HO‐1−/− mice were susceptible to oxidant stress, leading to cell death. Our data demonstrate that HO‐1 plays an essential protective role in the pathophysiology of atherosclerosis and vein graft stenosis.

Lipoxin A4 Regulates Natural Killer Cell and Type 2 Innate Lymphoid Cell Activation in Asthma

The pro-resolving mediator lipoxin A4 regulates natural killer cells and type 2 innate lymphoid cells in severe asthma. ILCs Catch Their Breath Focusing on your breath is one of the most basic meditation techniques. But for people with asthma, breathing can be anything but relaxing. Asthma is a chronic inflammatory disease, but attacks can be triggered by everything from allergens to cold to exercise. However, much still remains to be learned about the dysregulation that allows this inflammation to continue. Now, Barnig et al. suggest that differences in innate lymphoid cells (ILCs) may contribute to asthma pathogenesis. The authors look at two types of ILCs—natural killer (NK) cells and type 2 ILCs (ILC2s)—in the context of severe asthma. They find that NK cells may down-modulate the airway inflammatory response by inducing eosinophil apoptosis, whereas ILC2s promote airway inflammation through the secretion of interleukin-13 (IL-13). Intriguingly, despite their disparate function, both NK cells and ILC2s express the receptor for lipoxin A4, which functions in resolving inflammation. Indeed, lipoxin A4 increased the ability of NK cells to induce eosinophil apoptosis and decreased IL-13 production from the ILC2 population. Furthermore, lipoxin A4 production was decreased in severe asthmatics, suggesting this as a new line of potential therapies. Increasing lipoxin A4 may help severe asthmatics breathe easy. Asthma is a prevalent disease of chronic inflammation in which endogenous counterregulatory signaling pathways are dysregulated. Recent evidence suggests that innate lymphoid cells (ILCs), including natural killer (NK) cells and type 2 ILCs (ILC2s), can participate in the regulation of allergic airway responses, in particular airway mucosal inflammation. We have identified both NK cells and ILC2s in human lung and peripheral blood in healthy and asthmatic subjects. NK cells were highly activated in severe asthma, were linked to eosinophilia, and interacted with autologous eosinophils to promote their apoptosis. ILC2s generated antigen-independent interleukin-13 (IL-13) in response to the mast cell product prostaglandin D2 alone and in a synergistic manner with the airway epithelial cytokines IL-25 and IL-33. Both NK cells and ILC2s expressed the pro-resolving ALX/FPR2 receptors. Lipoxin A4, a natural pro-resolving ligand for ALX/FPR2 receptors, significantly increased NK cell–mediated eosinophil apoptosis and decreased IL-13 release by ILC2s. Together, these findings indicate that ILCs are targets for lipoxin A4 to decrease airway inflammation and mediate the catabasis of eosinophilic inflammation. Because lipoxin A4 generation is decreased in severe asthma, these findings also implicate unrestrained ILC activation in asthma pathobiology.

Inhibition of growth and p21ras methylation in vascular endothelial cells by homocysteine but not cysteine.

Although hyperhomocysteinemia has been recognized recently as a prevalent risk factor for myocardial infarction and stroke, the mechanisms by which it accelerates arteriosclerosis have not been elucidated, mostly because the biological effects of homocysteine can only be demonstrated at very high concentrations and can be mimicked by cysteine, which indicates a lack of specificity. We found that 10–50 μm of homocysteine (a range that overlaps levels observed clinically) but not cysteine inhibited DNA synthesis in vascular endothelial cells (VEC) and arrested their growth at the G1 phase of the cell cycle. Homocysteine in this same range had no effect on the growth of vascular smooth muscle cells (VSMC) or fibroblasts. Homocysteine decreased carboxyl methylation of p21 ras (a G1 regulator whose activity is regulated by prenylation and methylation in addition to GTP-GDP exchange) by 50% in VEC but not VSMC, a difference that may be explained by the ability of homocysteine to dramatically increase levels ofS-adenosylhomocysteine, a potent inhibitor of methyltransferase, in VEC but not VSMC. Moreover, homocysteine-induced hypomethylation in VEC was associated with a 66% reduction in membrane-associated p21 ras and a 67% reduction in extracellular signal-regulated kinase 1/2, which is a member of the mitogen-activated protein (MAP) kinase family. Because the MAP kinases have been implicated in cell growth, the p21 ras -MAP kinase pathway may represent one of the mechanisms that mediates homocysteine’s effect on VEC growth. VEC damage is a hallmark of arteriosclerosis. Homocysteine-induced inhibition of VEC growth may play an important role in this disease process.