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Angiogenesis in an in vivo model of adipose tissue development ¹

The Scripps Research Institute, Department of Cell Biology, Division of Vascular Biology, La Jolla, California, USA

² Correspondence: The Scripps Research Institute, Department of Cell Biology, Division of Vascular Biology, 10550 N. Torrey Pines Road, VB-3, La Jolla, CA 92037, USA. E-mail: loskutof{at}scripps.edu

SPECIFIC AIMS

Little is known about angiogenesis in adipose tissue, primarily because of the absence of reliable model systems. The general aim of these studies was to determine whether fat pads that develop when cultured 3T3-F442A cells are injected under the skin of nude mice form a suitable in vivo model system to study neovascularization of developing adipose tissue. One specific aim was to provide initial information about the morphology, kinetics of neovascularization, and gene expression profiles of the developing fat pads; another was to determine whether their new vasculature originated from bone marrow-derived endothelial progenitor cells or from preexisting host vessels.

PRINCIPAL FINDINGS

1. Fat pads formed by 3T3-F442A cells in nude mice are highly vascularized and morphologically similar to inguinal fat
Used to study adipogenesis, the 3T3-F442A in vivo fat pad model has not been used to examine adipose tissue neovascularization. In this model, 3T3-F442A preadipocytes are implanted subcutaneously into athymic Balb/c nude mice, where they develop into typical fat pads. Immunohistochemical staining for PECAM-1, a marker for endothelial cells (ECs), revealed the presence of microvessels in the developing fat pads 5 days after implantation. PECAM-1-positive neovasculature was detected throughout the developing fat pad by day 10; by day 21, morphology closely resembled that of control inguinal fat.

2. The in vivo fat pad model is well suited for gene profiling
We used quantitative real-time RT-PCR to monitor changes in angiogenesis- and adipogenesis-specific gene expression as a function of time. The expression of EC markers (PECAM-1, FLT-4, TIE-1, and TIE-2) and adipogenesis markers (lipoprotein lipase and adipsin) increase in parallel during fat pad development. Immunohistochemical staining for PECAM-1 demonstrated that the kinetics of angiogenesis in the fat pads paralleled the increase in formation of lipid-filled vacuoles in the cells, characteristic of adipogenesis. These observations suggest a close spatial relationship between blood vessel development and adipogenesis. Plasminogen activator inhibitor-1, tumor necrosis factor , and vascular endothelial growth factor A (VEGFA) were dramatically up-regulated during the first few days of fat pad development.

3. Origin of the neovasculature in the 3T3-F442A in vivo fat pad model: endothelial progenitor cells play little if any role
Experiments were performed to investigate the origin of the ECs that form the new vasculature in expanding adipose tissue. Three possibilities were considered: recruitment of endothelial progenitor cells (EPCs), conversion of the 3T3-F442A cells into ECs, and sprouting from existing host vessels.

To investigate the potential contribution of EPCs to adipose tissue development, fat pads were grown in athymic nude mice that had received a bone marrow transplantation from transgenic TIE2-GFP donor mice. Since these transgenic mice constitutively express GFP under transcriptional regulation of the EC-specific TIE-2 promoter, GFP expression should be restricted to the vasculature. The resulting fat pads were harvested, then stained for PECAM-1 to visualize the vasculature or for GFP to determine the degree of EPC incorporation. Although a PECAM-1-positive neovasculature was observed in developing fat pads from day 5 on, few GFP-positive cells were detected. The low number suggests these cells play little if any role in the neovascularization of developing fat pads.

4. Origin of the neovasculature in the 3T3-F442A in vivo fat pad model: the neovasculature is derived from the host and not from the injected 3T3-F442A cells
It is known that 3T3-L1 cells can express an endothelial phenotype under some conditions. The 3T3-F442A cell line used in our studies originated from the same parent cell line, raising the possibility that the neovasculature in the developing fat pads could be derived from the injected cells themselves. To investigate this, 3T3-F442A cells were labeled with a fluorescent green cell tracer stain and mixed with unlabeled cells, then injected into the mice. The resulting fat pads were harvested at various times, stained with the fluorescent EC-specific lectin, and analyzed by whole-mount laser confocal microscopy. If the neovasculature was derived from the injected cells, it should contain areas positive for both lectin (red) and cell tracer (green). Figure 1 A shows the border of a 1-wk-old fat pad. The lectin-stained vasculature appears to be invading a mix of labeled (green) and unlabeled injected 3T3-F442A cells. This neovasculature lacks detectable 3T3-F442A cells (green). The center of the 1-wk-old fat pad (Fig. 1B ) lacked a defined vasculature. By 10 days, the center of the fat pad (Fig. 1C ) contained a well-defined, lectin-positive vascular network along with individual green-labeled cells. However, there were no areas where the vasculature was positive for both the cell tracer and lectin; this was also true for 2-, 3-, and 4-wk-old fat pads. Thus, the vasculature appears to be derived from the host and not from the injected cells.

View larger version (61K): [in this window] [in a new window]   Figure 1. Behavior of injected 3T3-F442A cells in developing fat pads. Laser confocal images of whole mounts of fat pads that develop after injection of a 1:1 mix of 3 x 107 fluorescently labeled (green) with unlabeled 3T3-F442A cells. Tissues were stained with EC-specific fluorescent lectin (red). A) Border of a 1-wk-old fat pad; B) center of the same fat pad; C) the center of a 10-day-old fat pad. Scale bar: 50 µm.

5. Origin of the neovasculature in the 3T3-F442A in vivo fat pad model: the neovasculature originates by sprouting from larger, host-derived blood vessels that run parallel to peripheral nerves
Experiments were performed to determine whether angiogenesis (i.e., sprouting from preexisting blood vessels) is the mechanism of neovascularization in this model. Examination of a large number of fat pads by PECAM-1 staining revealed that although the fascia surrounding the fat pads varied in thickness, it remained largely devoid of vasculature at all times (see Fig. 2 B, C). Thus, the capillaries in the fat pads probably sprout from larger vessels that protrude through the fascia at select points: systematic analysis revealed the presence of such protruding vessels in a limited number of specific areas (arrows Fig. 2D-F ). These protruding vessels appear to branch from other large blood vessels outside the fascia that run parallel to nerve bundles (Fig. 2D-F ). Neurofilament (NF) staining shows that the fat pad is surrounded by nerve bundles (arrowheads, Fig. 2A ) associated with large blood vessels (representative example shown in Fig. 2G ). These blood vessel-associated nerve bundles reside just outside the fascia (Fig. 2A ) and likely are part of the sensory nervous system of the overlaying skin. These results suggest that the neovasculature in the developing fat pads is derived by sprouting from nerve-associated blood vessels.

View larger version (103K): [in this window] [in a new window]   Figure 2. PECAM-1 and NF expression in developing fat pads. A) Low-power overview of a fat pad stained for NF (brown); arrowheads indicate surrounding nerve bundles. B–F) Immunohistochemical staining for PECAM-1 at the borders of different fat pads illustrating the a-vascular fascia (B, C) and blood vessels that sprout from nerve-associated blood vessels and protrude (arrows) through the fascia (D–F). G) Higher magnification view of a blood vessel associated with two nerve bundles and stained for NF. F, fascia; M, muscle; N, nerve bundle; V, vessel. Scale bar: 100 µm (A); 50 µm (B–G).

CONCLUSIONS

We have shown that the 3T3-F442A in vivo model of adipose tissue development can be used to study angiogenesis. Besides being reproducible, the model was convenient for immunohistochemistry and gene profiling experiments; as illustrated in Fig. 3 , it could be used to study different inserts of fat pad development.

View larger version (29K): [in this window] [in a new window]   Figure 3. Schematic diagram illustrating the 3T3-F442A in vivo fat pad model and its potential to study neovascularization of developing adipose tissue. Immunohistochemistry and gene profiling analyses were used to determine the kinetics of fat pad neovascularization; results suggested a close spatial relationship between angiogenesis and adipogenesis. The neovasculature was shown to be derived from nerve-associated blood vessels from the host, and not from bone marrow-derived EPCs or from differentiation of the injected 3T3-F442A preadipocytes into ECs.

We demonstrated by immunohistochemistry and gene profiling a close spatial relationship between blood vessel development and adipogenesis. Cross-talk between adipocytes and ECs mediated by extracellular matrix components likely plays a role in coordinating angiogenesis and adipogenesis in the developing adipose tissue.

The unique characteristics of specific vascular beds depend on their anatomical location. Our observation that FLT-4 expression increases in parallel to that of PECAM-1, TIE-1, and TIE-2 was surprising, as this receptor had been thought to be a specific marker for lymphatic endothelia, and adipose tissue has been described to lack lymphatics. However, FLT-4 was shown to be expressed in many fenestrated endothelia, and adipose tissue was recently shown to contain fenestrated endothelia. The increase observed in FLT-4 raises the possibility that neovascularization of the developing fat pads involves fenestrated endothelia, usually present in capillaries of tissues where extensive molecular exchange occurs across the vessel wall (endocrine glands and the kidney). This function seems to be important for the adipose tissue since it releases a variety of regulatory molecules into the circulatory system.

It has become apparent that circulating bone marrow-derived EPCs are involved in promoting physiologic and pathologic neovascularization during wound healing and tumor growth, but questions remain about the magnitude of their contribution to newly forming blood vessels. EPCs were rarely detected in the fat pad neovasculature, suggesting they do not play a major role in the formation of new vessels in this model. However, EPCs contribute to neovascularization in other models, possibly reflecting the use of angiogenic growth factors. Other differences that may alter the recruitment and participation of circulating EPCs include variation in mechanical or biophysical properties inherent to a specific vascular locus, the absence or presence of inflammatory stimuli, and the unique microenvironment within different organ beds.

The new vessels that develop in 3T3-F422A-induced fat pads seem to originate predominantly by sprouting from larger blood vessels that run parallel to peripheral nerves. It was recently shown that arteries, but not veins, are aligned with peripheral nerves and that peripheral nerves seem to govern the pattern of blood vessel branching and arterial differentiation in the skin by the secretion of VEGF from sensory fibers and/or Schwann cells. These observations raise the possibility that nerves could guide the patterning of the blood vessels in the fat pads as well. This possibility seems unlikely since the neovasculature was observed as early as 5 days after cell implantation but the innervation of the fat pads was not evident until 6 weeks.

In summary, we have demonstrated that the 3T3-F442A in vivo fat pad model is well suited to study neovascularization of developing adipose tissue. We successfully used this model in gene profiling experiments and to study the origin of the neovasculature. This model should be useful in investigating the role of the extracellular matrix, fenestrated endothelia, and other factors in this process.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-1101fje;

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