Nancy R. Keller

Paracrine Regulation of Matrix Metalloproteinase Expression in the Normal Human Endometrium

Endometrial expression of matrix metalloproteinase (MMP)-3, MMP-7 and MMP-11 occurs during menstrual breakdown and subsequent estrogen-mediated growth, but not during the secretory phase. These enzymes are suppressed by progesterone treatment. Paracrine factors, including transforming growth factor-β (TGF-β) and retinoic acid, are also critical for MMP regulation in the endometrium. In contrast, inflammatory cytokines such as interleukin-1α may block or interfere with steroid-mediated MMP regulation at ectopic sites of growth. Using in vitro models, our laboratory has investigated the complex interactions between progesterone and locally produced cytokines that may affect MMP expression during the development of endometriosis. Our results indicate that targeting the regulation of MMPs may represent an appropriate therapeutic strategy for the treatment of endometriosis.

Portal Osmopressor Mechanism Linked to Transient Receptor Potential Vanilloid 4 and Blood Pressure Control

Human subjects with impaired baroreflex function cannot buffer rises or falls in blood pressure (BP), thus allowing BP effects of endogenous or environmental stimuli that previously escaped detection to emerge dramatically. Studies in these patients led us to discover that water ingestion induced a robust increase in BP and vascular resistance. Here, using a mouse model of baroreflex impairment, we show that the increase in blood pressure after water ingestion is mediated through sympathetic nervous system activation and that the osmosensitive transient receptor potential vanilloid 4 channel (Trpv4) is an essential component of the response. Although portal osmolality decreases after water ingestion in both wild-type and Trpv4−/− mice, only the wild-type animals show a pressor response. The same volume of physiological saline does not elicit an increase in BP, suggesting osmolality as the stimulus. The osmopressor response to water, and Trpv4 thus represent new factors now implicated in the physiology of BP regulation.

Norepinephrine Transporter–Deficient Mice Exhibit Excessive Tachycardia and Elevated Blood Pressure With Wakefulness and Activity

Background—Norepinephrine (NE) is a primary neurotransmitter of central autonomic regulation and sympathetic nerve conduction, and the norepinephrine transporter (NET) is crucial in limiting catecholaminergic signaling. NET is sensitive to antidepressants, cocaine, and amphetamine. NET blockade often is associated with cardiovascular side effects, and NET deficiency is linked to tachycardia in familial orthostatic intolerance. Methods and Results—We telemetrically monitored NET-deficient (NET−/−) mice to determine the cardiovascular effects of reduced NE reuptake. Mean arterial pressure was elevated in resting NET−/− mice compared with NET+/+ controls (103±0.6 versus 99±0.4 mm Hg; P<0.01), and corresponding pressures increased to 122±0.3 and 116±0.3 mm Hg (P<0.0001) with activity. Heart rate was also greater in resting NET−/− mice (565±5 versus 551±3 bpm; P<0.05), and genotypic differences were highly significant during the active phase (640±5 versus 607±3 bpm; P<0.0001). Conversely, the respiratory rate of resting NET−/− mice was dramatically reduced, whereas increases after the day/night shift surpassed those of controls. Plasma catecholamines in NET−/− and NET+/+ mice were as follows: NE, 69±8 and 32±7; dihydroxyphenylglycol, 2+0.4 and 17±3; epinephrine, 15±3 and 4±0.6; and dopamine, 13±4 and 4±1 pmol/mL. Catechols in urine, brain, and heart also were determined. Conclusions—Resting mean arterial pressure and heart rate are maintained at nearly normal levels in NET-deficient mice, most likely as a result of increased central sympathoinhibition. However, sympathetic activation with wakefulness and activity apparently overwhelms central modulation, amplifying peripheral catecholaminergic signaling, particularly in the heart.

Progesterone-Mediated Endometrial Maturation Limits Matrix Metalloproteinase (MMP) Expression in an Inflammatory-like Environment

Abstract: The human endometrium exhibits regular cycles of growth, differentiation, and breakdown in response to changing levels of ovarian steroids. Following the tissue loss and repair processes of menstruation, rising levels of estradiol initiate a development‐like process leading to a complete restructuring of the endometrial surface. In contrast, while under the predominate influence of progesterone, proliferation declines as cell‐specific differentiation prepares the endometrium for pregnancy over a 5‐ to 6‐day period. In the absence of nidation, steroid support is lost; the endometrial surface begins a complex process of tissue breakdown and bleeding, producing a viscous mixture of cellular debris within a bloody menstrual effluent. Although most of the menstrual fluid exits the body, reflux of some material occurs in most women, providing a poorly understood opportunity for ectopic endometrial growth and establishment of the disease endometriosis. The cyclic restructuring of the endometrium requires numerous matrix metalloproteinases (MMPs) that mediate normal and pathological tissue turnover throughout the reproductive tract. The expression of multiple MMPs facilitates degradation of extracellular matrix during growth‐related remodeling as well as tissue breakdown at the time of menstruation. However, these enzymes are absent during the early and midsecretory phase and the suppression of endometrial MMPs remains important to maintaining the integrity of the endometrium during the highly invasive events required to establish a normal hemochorial placenta. Several research groups have suggested that steroid‐mediated expression and action of MMPs during the menstrual cycle may provide a key mechanistic link between endometrial turnover and the invasive processes necessary for establishment of endometriosis.

Tachycardia, reduced vagal capacity, and age-dependent ventricular dysfunction arising from diminished expression of the presynaptic choline transporter

Healthy cardiovascular function relies on a balanced and responsive integration of noradrenergic and cholinergic innervation of the heart. High-affinity choline uptake by cholinergic terminals is pivotal for efficient ACh production and release. To date, the cardiovascular impact of diminished choline transporter (CHT) expression has not been directly examined, largely due to the transporters inaccessibility in vivo. Here, we describe findings from cardiovascular experiments using transgenic mice that bear a CHT genetic deficiency. Whereas CHT knockout (CHT(-/-)) mice exhibit early postnatal lethality, CHT heterozygous (CHT(+/-)) mice survive, grow, and reproduce normally and exhibit normal spontaneous behaviors. However, the CHT(+/-) mouse heart displays significantly reduced levels of high-affinity choline uptake accompanied by significantly reduced levels of ACh. Telemeterized recordings of cardiovascular function in these mice revealed tachycardia and hypertension at rest. After treadmill exercise, CHT(+/-) mice exhibited slower heart rate recovery, consistent with a diminished cholinergic reserve, a contention validated through direct vagal nerve stimulation. Echocardiographic and histological experiments revealed an age-dependent decrease in fractional shortening, increased left ventricular dimensions, and increased ventricular fibrosis, consistent with ventricular dysfunction. These cardiovascular phenotypes of CHT(+/-) mice encourage an evaluation of humans bearing reduced CHT expression for their resiliency in maintaining proper heart function as well as risk for cardiovascular disease.

Genetic basis of clinical catecholamine disorders

Abstract: Norepinephrine and epinephrine are critical determinants of minute‐to‐minute regulation of blood pressure. Here we review the characterization of two syndromes associated with a genetic abnormality in the noradrenergic pathway. In 1986, we reported a congenital syndrome of undetectable tissue and circulating levels of norepinephrine and epinephrine, elevated levels of dopamine, and absence of dopamine‐β‐hydroxylase (DBH). These patients appeared with ptosis and severe orthostatic hypotension and lacked sympathetic noradrenergic function. In two persons with DBH deficiency, we identified seven novel polymorphisms. Both patients are compound heterozygotes for a variant that affects expression of DBH protein via impairment of splicing. Patient 1 also has a missense mutation in DBH exon 2, and patient 2 carries missense mutations in exons 1 and 6. Orthostatic intolerance is a common syndrome affecting young women, presenting with orthostatic tachycardia and symptoms of cerebral hypoperfusion on standing. We tested the hypothesis that abnormal norepinephrine transporter (NET) function might contribute to its etiology. In our proband, we found an elevated plasma norepinephrine with standing that was disproportionate to the increase in levels of dihydroxphenylglycol, as well as impaired norepinephrine clearance and tyramine resistance. Studies of NET gene structure revealed a coding mutation converting a conserved alanine residue in transmembrane domain 9 to proline. Analysis of the protein produced by the mutant cDNA demonstrated greater than 98% reduction in activity relative to normal. The finding of genetic mutations responsible for DBH deficiency and orthostatic intolerance leads us to believe that genetic causes of other autonomic disorders will be found, enabling us to design more effective therapeutic interventions.

Gynecologic disorders and menstrual cycle lightheadedness in postural tachycardia syndrome

To assess differences in gynecologic history and lightheadedness during menstrual cycle phases among patients with POTS and healthy control women.

Familial orthostatic tachycardia.

Purpose of review Postural tachycardia syndrome is an autonomic disorder primarily of younger women. The patient population is heterogeneous, making diagnosis and treatment a challenge. A mutation in the norepinephrine (noradrenaline) transporter gene prompted further genetic analysis. Recent findings Eleven new mutations were found in the human norepinephrine transporter gene, although none were directly associated with postural tachycardia syndrome. The 5′-flanking −1012C → T variant of the dopamine β-hydroxylase gene was slightly increased and protection was associated with a reduced incidence of two mutations in the endothelial nitric oxide synthase gene, and one in endothelin-1. Mutations in other disease-related genes suggest a potential relationship with the pathogenesis of postural tachycardia syndrome. Benign joint hypermobility syndrome, for example, shares similar autonomic symptoms and is linked to a mutation in tenascin-X. Additional genetic findings are discussed as potential contributors to vascular health and neurodegeneration. Summary Genetic testing can reveal molecular mechanisms of disease and provide an additional strategy for diagnosis and treatment of heterogeneous patient populations such as postural tachycardia syndrome. It is quite likely that the pathogenesis of this disorder will be attributed to numerous genetic mutations, both subtle and overt. Therefore, continued study of the relationships between genotype and phenotype are necessary to better understand this syndrome and others with associated dysautonomia.

Mouse Homologous Recombination Models

Publisher Summary This chapter focuses on knockout and transgenic mice with an autonomic phenotype characterized by altered blood pressure, cardiac function, sympathetic nerve activity, or catecholamine levels. Disruption or overexpression of targeted mouse genes has been a boon to understanding the effect of individual genes in mammalian physiology. There has been an avalanche of reports describing historically well-recognized genes as well as a number of seemingly unrelated genes that have a significant impact on autonomic nervous system (ANS) function. Phenotypic analysis has revealed that embryonic knockouts are smaller in size with an enlarged right atrium, myocardial thinning, and overall myocardial hypoplasia. Decreased cardiac ejection was visualized with intravital microscopy in utero, suggesting heart failure as a probable cause of death with underlying ventricular chamber dysfunction and an associated thin myocardium syndrome. Phenotypic evaluation of animal models designed to mimic human behavior and psychiatric disorders have demonstrated that genetic makeup alone does not dictate complex functional changes and developmental adaptation, background strain, and environmental variables must also be considered when assigning phenotype.