Brown adipose tissue, because of its capacity for uncoupled mitochondrial respiration, has been implicated as an important site of facultative energy expenditure. This has led to speculation that this tissue normally functions to prevent obesity. Attempts to ablate or denervate brown adipose tissue surgically have been uninformative because it exists in diffuse depots and has substantial capacity for regeneration and hypertrophy. Here we have used a transgenic toxigene approach to create two lines of transgenic mice with primary deficiency of brown adipose tissue. At 16 days, both lines have decreased brown fat and obesity. In one line, brown fat subsequently regenerates and obesity resolves. In the other line, the deficiency persists and obesity, with its morbid complications, advances. Obesity develops in the absence of hyperphagia, indicating that brown fat deficient mice have increased metabolic efficiency. As obesity progresses, transgenic animals develop hyperphagia. This study supports a critical role for brown adipose tissue in the nutritional homeostasis of mice.

Phosphoinositide 3-kinase (PI3K) has been shown to regulate cell and organ size in Drosophila, but the role of PI3K in vertebrates in vivo is not well understood. To examine the role of PI3K in intact mammalian tissue, we have created and characterized transgenic mice expressing constitutively active or dominantnegative mutants of PI3K in the heart. Cardiacspeci®c expression of constitutively active PI3K resulted in mice with larger hearts, while dominantnegative PI3K resulted in mice with smaller hearts. The increase or decrease in heart size was associated with comparable increase or decrease in myocyte size. Cardiomyopathic changes, such as myocyte necrosis, apoptosis, interstitial ®brosis or contractile dysfunction, were not observed in either of the transgenic mice. Thus, the PI3K pathway is necessary and suf®-cient to promote organ growth in mammals.

TIE2 is a vascular endothelial-specific receptor tyrosine kinase essential for the regulation of vascular network formation and remodeling. Previously, we have shown that the 1.2-kb 5 flanking region of the TIE2 promoter is capable of directing ␤-galactosidase reporter gene expression specifically into a subset of endothelial cells (ECs) of transgenic mouse embryos. However, transgene activity was restricted to early embryonic stages and not detectable in adult mice. Herein we describe the identification and characterization of an autonomous endothelial-specific enhancer in the first intron of the mouse TIE2 gene. Furthermore, combination of the TIE2 promoter with an intron fragment containing this enhancer allows it to target reporter gene expression specifically and uniformly to virtually all vascular ECs throughout embryogenesis and adulthood. To our knowledge, this is the first time that an in vivo expression system has been assembled by which heterologous genes can be targeted exclusively to the ECs of the entire vasculature. This should be a valuable tool to address the function of genes during physiological and pathological processes of vascular ECs in vivo. Furthermore, we were able to identify a short region critical for enhancer function in vivo that contains putative binding sites for Ets-like transcription factors. This should, therefore, allow us to determine the molecular mechanisms underlying the vascular-EC-specific expression of the TIE2 gene.Establishment of the circulation is critical for the development of all organ systems of the human body, and endothelial cells (ECs), which line the lumina of all blood vessels, play an essential role in the formation and physiological functions of the circulatory system (for reviews, see refs. 1 and 2). In addition, the importance of the vascular system during pathological conditions such as cancer, atherosclerosis, and wound healing has also been well recognized (for review, see ref.3). However, little is known about the molecular mechanisms regulating the diverse functions of ECs in vivo.The recent development of transgenic mouse technology has proven to be a powerful tool for the examination of a number of mammalian developmental processes, and sequences that are able to drive gene expression in ECs of transgenic mice have been described (4-7). However, none of these sequences work uniformly in all ECs of all developmental stages or in the adult animal. Furthermore, some of these expression systems are not strictly EC-specific (5-7). Therefore, we have embarked on the development of an in vivo expression system in which heterologous gene expression can be targeted specifically and uniformly to ECs throughout development and the adult animal.The TIE2 gene, coding for one of the receptors of the newly identified family of regulators of vascular remodeling, called the angiopoietins (8, 9), appeared to be a promising candidate in the search for EC-specific transcriptional regulatory elements. This is because TIE2 expression becomes detectable as soo...

Vascular permeability factor/vascular endothelial growth factor (VPF/VEGF, VEGF-A) is a multifunctional cytokine with important roles in pathological angiogenesis. Using an adenoviral vector engineered to express murine VEGF-A164, we previously investigated the steps and mechanisms by which this cytokine induced the formation of new blood vessels in adult immunodeficient mice and demonstrated that the newly formed blood vessels closely resembled those found in VEGF-A–expressing tumors. We now report that, in addition to inducing angiogenesis, VEGF-A164 also induces a strong lymphangiogenic response. This finding was unanticipated because lymphangiogenesis has been thought to be mediated by other members of the VPF/VEGF family, namely, VEGF-C and VEGF-D. The new “giant” lymphatics generated by VEGF-A164 were structurally and functionally abnormal: greatly enlarged with incompetent valves, sluggish flow, and delayed lymph clearance. They closely resembled the large lymphatics found in lymphangiomas/lymphatic malformations, perhaps implicating VEGF-A in the pathogenesis of these lesions. Whereas the angiogenic response was maintained only as long as VEGF-A was expressed, giant lymphatics, once formed, became VEGF-A independent and persisted indefinitely, long after VEGF-A expression ceased. These findings raise the possibility that similar, abnormal lymphatics develop in other pathologies in which VEGF-A is overexpressed, e.g., malignant tumors and chronic inflammation.