A-type natriuretic peptide level in hypertensive transgenic mice.

A-type (atrial) natriuretic peptide (ANP) levels in heart and plasma were examined by immunohistochemistry, electron microscopy, and radioimmunoassay (RIA) in hypertensive transgenic mice (Tsukuba hypertensive mice; THM). Additionally, the ANP mRNA level in the heart was measured using real-time polymerase chain reaction (PCR) assay. The blood pressure and the ratio of heart weight to body weight in THM was significantly higher than those in the control mice (C57BL/6J). The number of ANP-granules and ANP immunoreactivity in the auricular cardiocytes were significantly lower in THM than in the control. Ultrastructurally, the ventricular cardiocytes in the THM occasionally had ANP-like granules, which were not present in the controls. Using RIA, the plasma, auricular, and ventricular ANP concentrations were significantly higher in THM than in the control, but there was no significant difference in plasma cyclic guanosine monophosphate (GMP) concentration between THM and the control. The ANP mRNA levels of the auricular and ventricular cardiocytes in the THM were siginificantly higher than those in the controls. The present study suggested that the ANP release system of the auricular cardiocytes in these transgenic mice is different from normal (control mice).     

Dose-dependent lung remodeling in transgenic mice expressing transforming growth factor-alpha

Transgenic mice overexpressing human transforming growth factor-alpha (TGF-alpha) develop emphysema and fibrosis during postnatal alveologenesis. To assess dose-related pulmonary alterations, four distinct transgenic lines expressing different amounts of TGF-alpha in the distal lung under control of the surfactant protein C (SP-C) promoter were characterized. Mean lung homogenate TGF-alpha levels ranged from 388 +/- 40 pg/ml in the lowest expressing line to 1,247 +/- 33 pg/ml in the highest expressing line. Histological assessment demonstrated progressive alveolar airspace size changes that were more severe in the higher expressing TGF-alpha lines. Pleural and parenchymal fibrosis were only detected in the highest expressing line (line 28), and increasing terminal airspace area was associated with increasing TGF-alpha expression. Hysteresis on pressure-volume curves was significantly reduced in line 28 mice compared with other lines of mice. There were no differences in bronchoalveolar lavage fluid cell count or differential that would indicate any evidence of lung inflammation among all transgenic lines. Proliferating cells were increased in line 28 without alterations of numbers of type II cells. We conclude that TGF-alpha lung remodeling in transgenic mice is dose dependent and is independent of pulmonary inflammation.     

Cardiac remodeling in erythropoietin-transgenic mice.

BACKGROUND: Transgenic (tg) mice with chronic overexpression of the human erythropoietin gene are characterized by an increased hematocrit of about 0.80 in adulthood. This is accompanied by cardiac dysfunction and premature death. The aim of this study was to examine whether this cardiac dysfunction was accompanied by hypertrophy of the heart with remodeling of the extracellular matrix (ECM). METHODS: 3-months-old wild type (wt) and tg mice without cardiac hypertrophy were compared with the respective 7-months-old mice. The mRNA of brain natriuretic peptide (BNP), of the matrix metalloproteinases (MMP)-2, -8, -9, -13, of the tissue inhibitor of metalloproteinase (TIMP)-1, -2, -3, -4 and of collagen I and III was detected by ribonuclease protection assay. The activity of MMPs was measured by zymography. RESULTS: There was hypertrophy of both ventricles in 7-months-old tg mice, which was accompanied by elevated mRNA expression of BNP. MMP-2 activity was increased and MMP-9 activity was decreased in the left ventricle (LV) of 3-months-old tg mice. This was accompanied by elevated TIMP-4 expression, followed by a shift of collagen mRNA expression from type III to type I in this ventricle. CONCLUSION: The shift to collagen I in the heart of tg mice might be associated with a stiffer ventricle resulting in diastolic dysfunction. This may be responsible for a relative and intermittent LV- and right ventricle (RV)-insufficiency which was likely to have occurred as evidenced by the elevation of lung and liver weight with hemorrhage and interstitial fibrosis after 7 months.     

Vascular remodeling in the circulations of the lung.

The lung is unique in its double sources of perfusion from the pulmonary and systemic circulations. One striking difference between the two circulations is the capacity for angiogenesis. The bronchial circulation has a capacity that seems quite similar to all systemic arteries, whereas the pulmonary circulation seems relatively inert in this regard. Extra-alveolar pulmonary arteries can grow somewhat in length, and septal capillaries seem to have the capability of reforming, but these processes do not seem to occur with nearly the same intensity associated with the bronchial arteries. In this review, we emphasize these differences between the two circulations of the lung, anticipating that future research will allow more focused probing into the molecular signaling that regulates the novel mechanistic and pathological pathways of each.     

The endothelin receptor antagonist ameliorates the hypertensive phenotypes of transgenic hypertensive mice with renin-angiotensin genes and discloses roles of organ specific activation of endothelin system in transgenic mice

Endothelin (ET)-1 and ET-2 are potent vasoconstrictor peptides with mitogenic activity. In this study, we investigated roles of ET system in renin-angiotensin system (RAS)-mediated hypertension, using transgenic hypertensive mice (THM) with over-expression of both human renin and angiotensinogen genes. In the first step, it was revealed that expression of ET system was locally enhanced, i.e. increases in cardiac preproET-1 mRNA and renal preproET-2 mRNA in THM, compared with the control (wild type) mice. In the next step, we studied the chronic effects of an ET antagonist (SB209670) on THM. Blood pressure (BP) in THM was significantly higher than that in the normal mice during the investigation. However, in the later phase of the study, from 12 to 20 weeks of treatment, THM receiving SB 209670 showed significantly lower BP than that in THM receiving saline. SB 209670 treatment for 20 weeks significantly attenuated phenotypes of cardiac hypertrophy, vascular wall thickening and hypertensive nephropathy observed in THM, suggesting that the ETA/B receptor antagonist is also effective even in the extraordinarily activated RAS condition. These findings suggest that organ specifically activated ET system in THM develops the phenotypes, hypertension, cardiac hypertrophy, and hypertensive nephropathy.     

Craniofacial dysmorphogenesis in transgenic mice.

While using transgenic mice to study the regulation of alpha-fetoprotein (AFP) it was noted that two different alpha-fetoprotein-chloramphenicol acetyl transferase (CAT) transgenes resulted in the appearance of craniofacial anomalies in 11% of the offspring derived from crosses between transgenic mice and nontransgenic mates. A total of 13 fetuses exhibited abnormalities; two are described in detail. Ninety-two percent of the affected fetuses had some form of mandibular abnormality while zygomatic and ossicular defects appeared in more than 40% of the specimens. Aglossia and aberrant musculature were also present in the most severely involved specimen. Eight of the affected fetuses were screened for the presence of the AFP-CAT plasmid and all were found to be heterozygous for the transgene. Since the probability that all 8 of the abnormal fetuses known to carry the CAT gene would have done so by chance was only 1 in 256, it may be assumed that these anomalies did not appear spontaneously, but were somehow created by the transgenic procedure. It is not known how the transgenic material led to the observed dysmorphogenetic pattern, but theoretically introduction of the AFP-CAT plasmid could have disrupted morphogenesis through the presence of the "foreign" CAT protein or a decrease in the availability of AFP. Since AFP levels were found to be normal in both the liver and the yolk sac of transgenic fetuses, it appears that the presence of CAT was responsible for the craniofacial anomalies described here.     

Cardiac myocyte apoptosis provokes adverse cardiac remodeling in transgenic mice with targeted TNF overexpression

Although cardiac myocyte apoptosis has been detected in explanted hearts from patients with end-stage dilated and ischemic cardiomyopathy, the relative contribution of apoptotic cell death to left ventricular (LV) remodeling and cardiac decompensation is not known. To determine whether progressive cardiac myocyte apoptosis contributes to the transition from a hypertrophic to a dilated cardiac phenotype that is observed in transgenic myosin heavy chain secreted TNF (MHCsTNF) mice with cardiac restricted overexpression of tumor necrosis factor (TNF), we assessed cardiac myocyte apoptosis (using a DNA ligase technique) in MHCsTNF mice and littermate control mice in relation to serial changes in LV structure, which was assessed using MRI. The prevalence of cardiac myocyte apoptosis increased progressively from 4 to 12 wk as the hearts of the MHCsTNF mice underwent the transition from a concentric hypertrophic to a dilated cardiac phenotype. Treatment of the MHCsTNF mice with the broad-based caspase inhibitor N-[(1,3-dimethylindole-2-carbonyl)-valinyl]-3-amino4-oxo-5-fluoropentanoic acid significantly decreased cardiac myocyte apoptosis and significantly attenuated LV wall thinning and adverse cardiac remodeling. Additional studies suggested that the TNF-induced decrease in Bcl-2 expression and activation of the intrinsic mitochondrial death pathway were responsible for the cardiac myocyte apoptosis observed in the MHCsTNF mice. These studies show that progressive cardiac myocyte apoptosis is sufficient to contribute to adverse cardiac remodeling in the adult mammalian heart through progressive LV wall thinning.     

Vascular remodeling by intussusceptive angiogenesis

Intussusception (growth within itself) is an alternative to the sprouting mode of angiogenesis. The protrusion of opposing microvascular walls into the capillary lumen creates a contact zone between endothelial cells. The endothelial bilayer is perforated, intercellular contacts are reorganized, and a transluminal pillar with an interstitial core is formed, which is soon invaded by myofibroblasts and pericytes leading to its rapid enlargement by the deposition of collagen fibrils. Intussusception has been implicated in three processes of vascular growth and remodeling. (1) Intussusceptive microvascular growth permits rapid expansion of the capillary plexus, furnishing a large endothelial surface for metabolic exchange. (2) Intussusceptive arborization causes changes in the size, position, and form of preferentially perfused capillary segments, creating a hierarchical tree. (3) Intussusceptive branching remodeling (IBR) leads to modification of the branching geometry of supplying vessels, optimizing pre- and postcapillary flow properties. IBR can also lead to the removal of branches by pruning in response to changes in metabolic needs. None of the three modes requires the immediate proliferation of endothelial cells but rather the rearrangement and plastic remodeling of existing ones. Intussusception appears to be triggered immediately after the formation of the primitive capillary plexus by vasculogenesis or sprouting. The advantage of this mechanism of growth over sprouting is that blood vessels are generated more rapidly in an energetically and metabolically more economic manner, as extensive cell proliferation, basement membrane degradation, and invasion of the surrounding tissue are not required; the capillaries thereby formed are less leaky. This process occurs without disrupting organ function. Improvements in our understanding of the process should enable the development of novel pro- and anti-angiogenic therapeutic treatments.Electronic Supplementary Material Supplementary material is available in the online version of this article at This research was supported by the Swiss National Science Foundation (grant no. 3100-055895.98/2) and the Bernese Cancer League     

Characterization of Pdgfrb-Cre transgenic mice reveals reduction of ROSA26 reporter activity in remodeling arteries

With the intention to modulate gene expression in vascular mural cells of remodeling vessels, we generated and characterized transgenic mouse lines with Cre recombinase under the control of the platelet-derived growth factor receptor-β promoter, referred to as Tg(Pdgfrb-Cre)(35Vli) . Transgenic mice were crossed with the Gt(ROSA)26Sor(tm1Sor) strain and examined for Cre activation by β-galactosidase activity, which was compared with endogenous Pdgfrb expression. In addition, Pdgfrb-Cre mice were used to drive expression of a conditional myc-tagged Cthrc1 transgene. There was good overlap of β-galactosidase activity with endogenous Pdgfrb immunoreactivity. However, dedifferentiation of vascular mural cells induced by carotid artery ligation revealed a dramatic discrepancy between ROSA26 reporter activity and Pdgfrb promoter driven Cre dependent myc-tagged Cthrc1 transgene expression. Our studies demonstrate the capability of the Pdgfrb-Cre mouse to drive conditional transgene expression as a result of prior Cre-mediated recombination in tissues known to express endogenous Pdgfrb. In addition, the study shows that ROSA26 promoter driven reporter mice are not suitable for lineage marking of smooth muscle in remodeling blood vessels.     

T cell autoimmunity in Ig transgenic mice.

Autoantibodies directed at a diverse group of proteins of the U1/Sm ribonucleoprotein (snRNP) are characteristic of systemic lupus erythematosus and are found in the MRL murine model of this disease. This study examines the role of transgenic B lymphocytes in the regulation of autoreactive T cells to the snRNP autoantigen. Transgenic mice were developed bearing an Ig heavy chain gene specific for the D protein component of murine snRNP. B lymphocytes in these mice are neither deleted nor anergic and are of an immature (heat-stable Aghigh) phenotype. T lymphocytes from anti-snRNP transgenic mice were examined using a recombinant form of the D protein of the murine snRNP complex. Our results revealed that transgenic anti-snRNP B cell APCs stimulated CD4 T cells from wild-type C57BL/6 and MRL lpr/lpr mice, while nonspecific APCs failed to stimulate CD4 T cells. This study demonstrates that autoreactive T cells are not deleted from wild-type mice, although their activation is facilitated by autoantigen-specific APCs. The snRNP-reactive T cells in C57BL/6 transgenic mice are tolerized, in contrast to those T cells from MRL lpr/lpr transgenic mice. These studies implicate a role for autoreactive B lymphocytes in the in vivo activation and/or diversification of autoreactive T cells.     

Human immunodeficiency virus-associated vasculopathy in transgenic mice.

There is substantial clinical evidence for the development of vascular disorders in human immunodeficiency virus (HIV)-infected individuals, particularly in the form of vasculitis. Transgenic mice carrying a replication-defective HIV-1 provirus with selective deletion of the gag, pol, and env genes developed extensive vasculopathy. Restricted expression of HIV nonstructural genes in smooth muscle cells was accompanied by the migration and proliferation of these cells in blood vessels of all sizes and at different body sites. The frequent infiltration observed in the hypertrophic vessel walls occurred predominantly in the adventitia and was composed of primarily T cells and occasionally plasma cells. The intimal thickening generated significant luminal narrowing in some vessels, and the restricted blood flow led to ischemia in the affected tissues. Interestingly, the endothelium did not appear to support HIV gene expression or be involved in the pathological process. This transgenic model provides an opportunity to dissect the mechanism underlying HIV-associated vasculopathy.     

The Hox gene family in transgenic mice.

Evidence is accumulating which suggests that the vertebrate Hox homeobox gene family plays an important role in pattern formation, particularly in the specification of regional diversity. In the last year important advances in the understanding of their regulation and function have been provided using transgenic mice.     

Nuclear remodeling and reprogramming in transgenic pig production

The manufacture of pigs with modifications to specific chromosomal regions requires that the modification first be made in somatic cells. The modified cells can then be used as donors for nuclear transfer (NT) in an attempt to clone that cell into a newborn animal. Unfortunately the procedures are inefficient and sometimes lead to animals that are abnormal. The cause of these abnormalities is likely established during the first cell cycle after the NT. Either the donor cell was abnormal or the oocyte cytoplasm was unable to adequately remodel the donor nucleus such that it was structured similar to the pronucleus of a zygote. A better understanding of chromatin remodeling and subsequent developmental gene expression will provide clues as to how procedures can be modified to generate fertile animals more efficiently.     

Vascular growth and remodeling in compensatory lung growth following right lobectomy.

Studies in animal models have shown that, following lobectomy (LBX), there is compensatory growth in the remaining lung. The vascular growth response following right LBX (R-LBX) is poorly understood. To test the hypothesis that arterial growth and remodeling occur in response to LBX, in proportion to the amount of right lung tissue removed, two (24% of lung mass; R-LBX2 group) or three right lobes (52% of lung mass; R-LBX3 group) were removed via thoracotomy from adult rats. Sham control animals underwent thoracotomy only. Arteriograms were generated 3 wk after surgery. The areas of the left lung arteriogram, arterial branching, length of arterial branches, arterial density, and arterial-to-alveolar ratios were measured. To determine whether R-LBX causes vascular remodeling and pulmonary hypertension, muscularization of arterioles and right ventricular hypertrophy were assessed. Lung weight and volume indexes were greater in R-LBX3. Arterial area of the left lung increased 26% in R-LBX2 and 47% in R-LBX3. The length of large arteries increased in R-LBX3 and to a lesser extent in R-LBX2. The ratio of distal pulmonary arteries to alveoli was similar after R-LBX2 compared with sham but was 30% lower in R-LBX3. Muscularization of arterioles increased after R-LBX3, but not in R-LBX2. Right ventricular hypertrophy increased 50-70% in R-LBX3, but not in R-LBX2. Whereas removal of three right lung lobes induced arterial growth in the left lungs of adult rats, which was proportionate to the number of lobes removed, the ratio of distal pulmonary arteries to alveoli was not normal, and vascular remodeling and pulmonary hypertension developed.     

Vascular remodeling after laser photocoagulation: concurrent regeneration and atrophy.

Laser photocoagulation is associated with paradoxical results: it causes obliteration of vessels, but leads also to the formation of new ones. In an attempt to better understand this dual vascular response we conducted an ultrastructure study of the choroidal vascular repair following krypton laser injury in rats. Three processes were observed: recanalization, neovascularization, and atrophy of both recanalized and newly formed capillaries. Post-lasering repair of the choroidal vasculature can therefore be described as a remodeling process, characterized by both regeneration and involution. The latter appears to be a secondary process of atrophy, contributing to permanent vascular obliteration. These mechanisms might explain why, in spite of initial vascular regeneration, laser photocoagulation treatment has a beneficial effect on choroidal subretinal neovascularization.     

High-frequency germ line gene conversion in transgenic mice.

Gene conversion is the nonreciprocal transfer of genetic information between two related genes or DNA sequences. It can influence the evolution of gene families, having the capacity to generate both diversity and homogeneity. The potential evolutionary significance of this process is directly related to its frequency in the germ line. While measurement of meiotic inter- and intrachromosomal gene conversion frequency is routine in fungal systems, it has hitherto been impractical in mammals. We have designed a system for identifying and quantitating germ line gene conversion in mice by analyzing transgenic male gametes for a contrived recombination event. Spermatids which undergo the designed intrachromosomal gene conversion produce functional beta-galactosidase (encoded by the lacZ gene), which is visualized by histochemical staining. We observed a high incidence of lacZ-positive spermatids (approximately 2%), which were produced by a combination of meiotic and mitotic conversion events. These results demonstrate that gene conversion in mice is an active recombinational process leading to nonparental gametic haplotypes. This high frequency of intrachromosomal gene conversion seems incompatible with the evolutionary divergence of newly duplicated genes. Hence, a process may exist to uncouple gene pairs from frequent conversion-mediated homogenization.     

Antiviral immune responses in CTLA4 transgenic mice.

The role of B7 binding CD28 in the regulation of T- and B-cell responses against viral antigens was assessed in transgenic mice expressing soluble CTLA4-Hgamma1 (CTLA4-Ig tg mice) that blocks B7-CD28 interactions. The results indicate that transgenic soluble CTLA4 does not significantly alter cytotoxic T-cell responses against replicating lymphocytic choriomeningitis virus (LCMV) or vaccinia virus but drastically impairs the induction of cytotoxic T-cell responses against abortively replicating vesicular stomatitis virus (VSV). While the T-independent neutralizing immunoglobulin M (IgM) responses were within normal ranges, the switch to IgG was reduced 4- to 16-fold after immunization with abortively replicating VSV and more than 30-fold after immunization with an inert VSV glycoprotein antigen in transgenic mice. IgG antibody responses to LCMV, as detected by enzyme-linked immunosorbent assay and by neutralizing action, were reduced about 3- to 20-fold and more than 50-fold, respectively. These results suggest that responses in CTLA4-Ig tg mice are mounted according to their independence of T help. While immune responses to nonreplicating or poorly replicating antigens are in general most dependent on T help and B7-CD28 interactions, they are most impaired in CTLA4-Ig tg mice. The results of the present experiments also indicate that highly replicating viruses, because of greater quantities of available antigens and by inducing as-yet-undefined factors and/or cell surface changes, are capable of compensating for the decrease in T help caused by the blocking effects of soluble CTLA4.