Ultrasound-Guided Intrauterine Labeling of Rat Fetuses.

AIM: To compare intra-amniotic versus fetal subcutaneous injections for selective fetal labeling in multifetal rat pregnancies. METHODS: A total of 14 pregnant rats were randomized to receive intra-amniotic injections of dyes (including Fluorescein, Indigo Carmine, or Evans Blue) or fetal subcutaneous injections (of commercial tattoo ink) both guided by ultrasound at 15-17 days of gestation. Survival, injection, and labeling success rates of both techniques were compared. RESULTS: Survival rates (84.4% for intra-amniotic injections vs. 90.9% for fetal subcutaneous injections) and injection success rates (94% for intra-amniotic injections vs. 100% for fetal subcutaneous injections) were similar among both groups. None of the neonates from the intra-amniotic injections group were labeled at birth, while 93% of the neonates from fetal subcutaneous injections group were tagged, showing a visible spot in the skin at birth. CONCLUSION: Our results suggest that ultrasound-guided fetal subcutaneous injections might be an adequate strategy for selectively labeling fetuses in multifetal pregnant animals.

Targeting repulsive guidance molecule A to promote regeneration and neuroprotection in multiple sclerosis.

Repulsive guidance molecule A (RGMa) is a potent inhibitor of neuronal regeneration and a regulator of cell death, and it plays a role in multiple sclerosis (MS). In autopsy material from progressive MS patients, RGMa was found in active and chronic lesions, as well as in normal-appearing gray and white matter, and was expressed by cellular meningeal infiltrates. Levels of soluble RGMa in the cerebrospinal fluid were decreased in progressive MS patients successfully treated with intrathecal corticosteroid triamcinolone acetonide (TCA), showing functional improvements. In vitro, RGMa monoclonal antibodies (mAbs) reversed RGMa-mediated neurite outgrowth inhibition and chemorepulsion. In animal models of CNS damage and MS, RGMa antibody stimulated regeneration and remyelination of damaged nerve fibers, accelerated functional recovery, and protected the retinal nerve fiber layer as measured by clinically relevant optic coherence tomography. These data suggest that targeting RGMa is a promising strategy to improve functional recovery in MS patients.

Postsystolic shortening by myocardial deformation imaging as a sign of cardiac adaptation to pressure overload in fetal growth restriction.

BACKGROUND: Fetal growth restriction (FGR) is associated with global adverse cardiac remodeling in utero and increased cardiovascular mortality in adulthood. Prenatal myocardial deformation has not been evaluated in FGR to date. We aimed to evaluate prenatal cardiac remodeling comprehensively in FGR including myocardial deformation imaging. METHODS AND RESULTS: Echocardiography was performed in 37 consecutive FGR (defined as birthweight <10th centile) and 37 normally grown fetuses. A comprehensive fetal echocardiography was performed including tissue Doppler and 2-dimensional-derived strain and strain rate. Postnatal blood pressure measurement at 6 months of age was also performed. FGR cases showed signs of more globular hearts with decreased longitudinal motion (left systolic annular peak velocity: controls mean 6 cm/s [SD 1.2] versus FGR 5.3 [1]) and diastolic dysfunction (isovolumic relaxation time: controls 44 ms [6] versus FGR 52 [9]). Peak strain and strain rate values of the left ventricle were not significantly different; however, a postsystolic shortening in the basal segment of the septal ventricular wall was observed in 57% of the FGR cases and in none of controls (P<0.001). FGR cases with postsystolic shortening had absence of a hypertrophic response, a poorer perinatal outcome (lower gestational age and birthweight, containing all cases of perinatal mortality [8%]), and higher values of blood pressure. CONCLUSIONS: Myocardial deformation imaging revealed a postsystolic shortening in 57% of FGR, which supports increased pressure overload as a mechanism for cardiovascular programming in FGR. Postsystolic shortening was associated with severity and with higher blood pressure postnatally.

Long-term follow-up of intrauterine growth restriction: cardiovascular disorders.

In the modern world, cardiovascular disorders are the leading cause of mortality in developed countries, which in most cases undergo a long subclinical phase that can last decades before the first clinical symptoms appear. Aside from the well-known risk factors related to lifestyle and genetics, there is growing evidence that in a proportion of cases, the predisposition to cardiovascular disease lies in prenatal life. Moreover, numerous historical cohort studies and animal models have shown a clear association between low birth weight and increased cardiovascular mortality in adulthood, including increased risk of hypertension, diabetes, dyslipidemia and coagulation disorders in children and adults. Besides premature birth, low birth weight in the majority of the cases is caused by intrauterine growth restriction (IUGR), which affects up to 10% of all births. Several clinical and experimental studies showed that IUGR fetuses present signs of cardiac dysfunction in utero that persist postnatally and may condition higher cardiovascular risk later in life. The present review discusses the importance of the long-term cardiovascular follow-up of the patients who suffered early or late IUGR in utero, particularly with regard to the long-term epidemiological studies in adults, prospective studies in children and the possible mechanisms that trigger IUGR and cardiovascular programming. Considering the high prevalence of IUGR and the progressing availability of intervention strategies, it is of the highest clinical relevance to detect cardiovascular risks as early as possible, to introduce timely preventive interventions and to adapt the lifestyle, in order to improve the long-term cardiovascular health outcome of IUGR cases.

Junb regulates arterial contraction capacity, cellular contractility, and motility via its target Myl9 in mice.

Cellular contractility and, thus, the ability to alter cell shape are prerequisites for a number of important biological processes such as cytokinesis, movement, differentiation, and substrate adherence. The contractile capacity of vascular smooth muscle cells (VSMCs) is pivotal for the regulation of vascular tone and thus blood pressure and flow. Here, we report that conditional ablation of the transcriptional regulator Junb results in impaired arterial contractility in vivo and in vitro. This was exemplified by resistance of Junb-deficient mice to DOCA-salt-induced volume-dependent hypertension as well as by a decreased contractile capacity of isolated arteries. Detailed analyses of Junb-deficient VSMCs, mouse embryonic fibroblasts, and endothelial cells revealed a general failure in stress fiber formation and impaired cellular motility. Concomitantly, we identified myosin regulatory light chain 9 (Myl9), which is critically involved in actomyosin contractility and stress fiber assembly, as a Junb target. Consistent with these findings, reexpression of either Junb or Myl9 in Junb-deficient cells restored stress fiber formation, cellular motility, and contractile capacity. Our data establish a molecular link between the activator protein-1 transcription factor subunit Junb and actomyosin-based cellular motility as well as cellular and vascular contractility by governing Myl9 transcription.

Upregulation of glutathione peroxidase offsets stretch-induced proatherogenic gene expression in human endothelial cells.

OBJECTIVE: Localization of atherosclerotic plaques typically correlates with areas of biomechanical strain where shear stress is decreased while stretch, thought to promote atherogenesis through enhanced oxidative stress, is increased. METHODS AND RESULTS: In human cultured endothelial cells, nitric oxide synthase expression was exclusively shear stress-dependent whereas expression of glutathione peroxidase-1 (GPx-1), but not that of Cu(2+)/Zn(2+)-superoxide dismutase or Mn(2+)-superoxide dismutase, was upregulated solely in response to cyclic stretch. GPx-1 expression was also enhanced in isolated mouse arteries perfused at high pressure. Combined pharmacological and decoy oligodeoxynucleotide blockade revealed that activation of p38 MAP kinase followed by nuclear translocation of CCAAT/enhancer binding protein plays a pivotal role in stretch-induced GPx-1 expression in human endothelial cells. Antisense oligodeoxynucleotide knockdown of GPx-1 reinforced both their capacity to generate hydrogen peroxide and the transient stretch-induced expression of CD40, monocyte chemoatractant protein-1, and vascular cell adhesion molecule-1. Consequently, THP-1 monocyte adhesion to the GPx-1-depleted cells was augmented. CONCLUSIONS: Stretch-induced proatherosclerotic gene expression in human endothelial cells seems to be hydrogen peroxide-mediated. The concomitant rise in GPx-1 expression, but not that of other antioxidant enzymes, may comprise an adaptive mechanism through which the cells maintain their antiatherosclerotic properties in spite of a decreased bioavailability of nitric oxide.

Stretch-induced activation of the transcription factor activator protein-1 controls monocyte chemoattractant protein-1 expression during arteriogenesis.

Cerebral, coronary, and peripheral artery diseases combined represent the most frequent cause of death in developed nations. The underlying progressive occlusion of large conductance arteries can partially be compensated for by transformation of preexisting collateral arterioles to small artery bypasses, a process referred to as arteriogenesis. Because biomechanical forces have been implicated in the initiation of arteriogenesis, we have investigated the mechanosensitive expression of a pivotal proarteriogenic molecule, monocyte chemoattractant protein (MCP)-1, which governs the recruitment of circulating monocytes to the wall of the remodeling collateral arterioles. Using a new ear artery ligation model and the classic hindlimb ischemia model in mice, we noted that MCP-1 expression is significantly increased in collateral arterioles undergoing arteriogenesis already 24 hours after its onset. By mimicking proarteriogenic perfusion conditions in small mouse arteries, we observed that MCP-1 expression is predominantly upregulated in the smooth muscle cells, which solely sense changes in circumferential wall tension or stretch. Subsequent analyses of cultured endothelial and smooth muscle cells confirmed that cyclic stretch but not shear stress upregulates MCP-1 expression in these cells. Blockade of the mechanosensitive transcription factor activator protein-1 by using a specific decoy oligodeoxynucleotide abolished this stretch-induced MCP-1 expression. Likewise, topical administration of the decoy oligodeoxynucleotide to the mouse ear abrogated arteriogenesis through downregulation of MCP-1 expression and monocyte recruitment. Collectively, these findings point toward a stretch-induced activator protein-1-mediated rise in MCP-1 expression in vascular smooth muscle cells as a critical determinant for the initiation of arteriogenesis.