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Mechanisms of lymphatic regeneration after tissue transfer.

Yan A, Avraham T, Zampell JC, Aschen SZ, Mehrara BJ - PLoS ONE (2011)

Bottom Line: Patterns of VEGF-C expression and macrophage infiltration were temporally and spatially associated with lymphatic regeneration.When compared to mice treated with excision only, there was a 4-fold decrease in tail volumes, 2.5-fold increase in lymphatic transport by lymphoscintigraphy, 40% decrease in dermal thickness, and 54% decrease in scar index in skin-grafted animals, indicating that tissue transfer could bypass damaged lymphatics and promote rapid lymphatic regeneration.This process is temporally and spatially associated with VEGF-C expression and macrophage infiltration.

View Article: PubMed Central - PubMed

Affiliation: The Division of Plastic and Reconstructive Surgery, Department of Surgery, Memorial Sloan-Kettering Cancer Center, New York, New York, United States of America.

ABSTRACT

Introduction: Lymphedema is the chronic swelling of an extremity that occurs commonly after lymph node resection for cancer treatment. Recent studies have demonstrated that transfer of healthy tissues can be used as a means of bypassing damaged lymphatics and ameliorating lymphedema. The purpose of these studies was to investigate the mechanisms that regulate lymphatic regeneration after tissue transfer.

Methods: Nude mice (recipients) underwent 2-mm tail skin excisions that were either left open or repaired with full-thickness skin grafts harvested from donor transgenic mice that expressed green fluorescent protein in all tissues or from LYVE-1 knockout mice. Lymphatic regeneration, expression of VEGF-C, macrophage infiltration, and potential for skin grafting to bypass damaged lymphatics were assessed.

Results: Skin grafts healed rapidly and restored lymphatic flow. Lymphatic regeneration occurred beginning at the peripheral edges of the graft, primarily from ingrowth of new lymphatic vessels originating from the recipient mouse. In addition, donor lymphatic vessels appeared to spontaneously re-anastomose with recipient vessels. Patterns of VEGF-C expression and macrophage infiltration were temporally and spatially associated with lymphatic regeneration. When compared to mice treated with excision only, there was a 4-fold decrease in tail volumes, 2.5-fold increase in lymphatic transport by lymphoscintigraphy, 40% decrease in dermal thickness, and 54% decrease in scar index in skin-grafted animals, indicating that tissue transfer could bypass damaged lymphatics and promote rapid lymphatic regeneration.

Conclusions: Our studies suggest that lymphatic regeneration after tissue transfer occurs by ingrowth of lymphatic vessels and spontaneous re-connection of existing lymphatics. This process is temporally and spatially associated with VEGF-C expression and macrophage infiltration. Finally, tissue transfer can be used to bypass damaged lymphatics and promote rapid lymphatic regeneration.

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Related in: MedlinePlus

Autogenous tissue lymphangiogenesis is associated with spontaneous reconnection of local lymphatics and infiltration of new lymphatic capillaries.A. Skin grafts harvested from GFP transgenic mice express GFP at high levels and for a long period of time. A representative fluorescent picture of a mouse (top) with higher magnification of the tail (bottom) is shown 6 weeks after surgery. The skin-grafted GFP portion of the tail can easily be seen. B. Gross photographs of mouse tails treated with skin grafts harvested from GFP transgenic mice. Note the rapid incorporation and ingrowth of hair follicles at the 6-week time point. C. Microlymphangiography performed 2 (left) or 6 (right) weeks following skin graft. Distal portion of the tail is shown at the bottom. Note the flow of fluorescent dye by interstitial flow after 2 weeks. In contrast, lymphatic flow can be seen in the skin-grafted area at the 6-week time point. Representative figures of triplicate experiments are shown. D. Higher power photograph of microlymphangiography 2 and 6 weeks after surgery demonstrating a few honeycomb-like dermal lymphatics (white arrows) in the skin graft at the 6-week time point. E. Representative LYVE-1 (pink) and GFP (green) co-localization in skin-grafted mouse tails 6 weeks after surgery. Low power (5x; left) and high power (20x; right) views are shown. Note connection between GFP- (recipient) and GFP+ (donor) lymphatic vessel. F. Representative photomicrograph (2x) demonstrating GFP (left), LYVE-1 (middle), and co-localization (right) of skin-grafted mouse tails 6 weeks after surgery. Note ingrowth of GFP-/LYVE-1+ vessels (yellow circle) from the distal (yellow dotted line) portion of the wound. G. High power (20x) view of section shown in F. Note the presence of both recipient (GFP-/LYVE-1+) and donor (GFP+/LYVE-1+) lymphatics at the distal margin of the wound.
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pone-0017201-g001: Autogenous tissue lymphangiogenesis is associated with spontaneous reconnection of local lymphatics and infiltration of new lymphatic capillaries.A. Skin grafts harvested from GFP transgenic mice express GFP at high levels and for a long period of time. A representative fluorescent picture of a mouse (top) with higher magnification of the tail (bottom) is shown 6 weeks after surgery. The skin-grafted GFP portion of the tail can easily be seen. B. Gross photographs of mouse tails treated with skin grafts harvested from GFP transgenic mice. Note the rapid incorporation and ingrowth of hair follicles at the 6-week time point. C. Microlymphangiography performed 2 (left) or 6 (right) weeks following skin graft. Distal portion of the tail is shown at the bottom. Note the flow of fluorescent dye by interstitial flow after 2 weeks. In contrast, lymphatic flow can be seen in the skin-grafted area at the 6-week time point. Representative figures of triplicate experiments are shown. D. Higher power photograph of microlymphangiography 2 and 6 weeks after surgery demonstrating a few honeycomb-like dermal lymphatics (white arrows) in the skin graft at the 6-week time point. E. Representative LYVE-1 (pink) and GFP (green) co-localization in skin-grafted mouse tails 6 weeks after surgery. Low power (5x; left) and high power (20x; right) views are shown. Note connection between GFP- (recipient) and GFP+ (donor) lymphatic vessel. F. Representative photomicrograph (2x) demonstrating GFP (left), LYVE-1 (middle), and co-localization (right) of skin-grafted mouse tails 6 weeks after surgery. Note ingrowth of GFP-/LYVE-1+ vessels (yellow circle) from the distal (yellow dotted line) portion of the wound. G. High power (20x) view of section shown in F. Note the presence of both recipient (GFP-/LYVE-1+) and donor (GFP+/LYVE-1+) lymphatics at the distal margin of the wound.

Mentions: In order to investigate the involvement of recipient and donor cells in spontaneous lymphatic regeneration, the tails of athymic nude mice that underwent full-thickness skin excision were repaired with skin grafts harvested from GFP mice. This allowed us to follow the origin of cells by co-localization of GFP and lymphatic markers. Using this technique, the recipient nude mice lymphatics stained positive for lymphatic markers but negative for GFP. The transplanted tissue lymphatics, in contrast, stained positive for both GFP and lymphatic markers (Figure 1). Using fluorescent imaging, we found that high-level GFP expression was maintained in the transplanted skin even 6 weeks after tissue transfer (Figure 1A). The transplanted skin grafts healed quickly and with regrowth of hair and skin appendages by 6 weeks indicating that the grafts had become fully incorporated (Figure 1B). In addition, return of lymphatic function with transport of fluorescence-labeled colloid could be grossly visualized by microlymphangiography as early as 2 weeks after surgery (Figure 1C). This transport was initially driven by interstitial flow (i.e. no discrete lymphatic vessels could be visualized at 2 weeks); however, by 6 weeks honeycomb-like dermal lymphatics were noted in the transferred grafts (primarily at the distal margin) suggesting that the reformed lymphatic vessels are functional (Figure 1D). The lack of complete restoration of the honeycomb-like dermal lymphatic architecture in the mid portion of the graft at this time may reflect a delay in dermal lymphatic regeneration. This concept is supported by previous studies demonstrating that honeycomb-like lymphatic vessels that regenerate in the mouse tail after coverage of the wound with collagen gel are not ordinarily visible until day 60 after surgery and even at this time are only seen at the distal margin of the wound.[21], [22]


Mechanisms of lymphatic regeneration after tissue transfer.

Yan A, Avraham T, Zampell JC, Aschen SZ, Mehrara BJ - PLoS ONE (2011)

Autogenous tissue lymphangiogenesis is associated with spontaneous reconnection of local lymphatics and infiltration of new lymphatic capillaries.A. Skin grafts harvested from GFP transgenic mice express GFP at high levels and for a long period of time. A representative fluorescent picture of a mouse (top) with higher magnification of the tail (bottom) is shown 6 weeks after surgery. The skin-grafted GFP portion of the tail can easily be seen. B. Gross photographs of mouse tails treated with skin grafts harvested from GFP transgenic mice. Note the rapid incorporation and ingrowth of hair follicles at the 6-week time point. C. Microlymphangiography performed 2 (left) or 6 (right) weeks following skin graft. Distal portion of the tail is shown at the bottom. Note the flow of fluorescent dye by interstitial flow after 2 weeks. In contrast, lymphatic flow can be seen in the skin-grafted area at the 6-week time point. Representative figures of triplicate experiments are shown. D. Higher power photograph of microlymphangiography 2 and 6 weeks after surgery demonstrating a few honeycomb-like dermal lymphatics (white arrows) in the skin graft at the 6-week time point. E. Representative LYVE-1 (pink) and GFP (green) co-localization in skin-grafted mouse tails 6 weeks after surgery. Low power (5x; left) and high power (20x; right) views are shown. Note connection between GFP- (recipient) and GFP+ (donor) lymphatic vessel. F. Representative photomicrograph (2x) demonstrating GFP (left), LYVE-1 (middle), and co-localization (right) of skin-grafted mouse tails 6 weeks after surgery. Note ingrowth of GFP-/LYVE-1+ vessels (yellow circle) from the distal (yellow dotted line) portion of the wound. G. High power (20x) view of section shown in F. Note the presence of both recipient (GFP-/LYVE-1+) and donor (GFP+/LYVE-1+) lymphatics at the distal margin of the wound.
© Copyright Policy
Related In: Results  -  Collection

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pone-0017201-g001: Autogenous tissue lymphangiogenesis is associated with spontaneous reconnection of local lymphatics and infiltration of new lymphatic capillaries.A. Skin grafts harvested from GFP transgenic mice express GFP at high levels and for a long period of time. A representative fluorescent picture of a mouse (top) with higher magnification of the tail (bottom) is shown 6 weeks after surgery. The skin-grafted GFP portion of the tail can easily be seen. B. Gross photographs of mouse tails treated with skin grafts harvested from GFP transgenic mice. Note the rapid incorporation and ingrowth of hair follicles at the 6-week time point. C. Microlymphangiography performed 2 (left) or 6 (right) weeks following skin graft. Distal portion of the tail is shown at the bottom. Note the flow of fluorescent dye by interstitial flow after 2 weeks. In contrast, lymphatic flow can be seen in the skin-grafted area at the 6-week time point. Representative figures of triplicate experiments are shown. D. Higher power photograph of microlymphangiography 2 and 6 weeks after surgery demonstrating a few honeycomb-like dermal lymphatics (white arrows) in the skin graft at the 6-week time point. E. Representative LYVE-1 (pink) and GFP (green) co-localization in skin-grafted mouse tails 6 weeks after surgery. Low power (5x; left) and high power (20x; right) views are shown. Note connection between GFP- (recipient) and GFP+ (donor) lymphatic vessel. F. Representative photomicrograph (2x) demonstrating GFP (left), LYVE-1 (middle), and co-localization (right) of skin-grafted mouse tails 6 weeks after surgery. Note ingrowth of GFP-/LYVE-1+ vessels (yellow circle) from the distal (yellow dotted line) portion of the wound. G. High power (20x) view of section shown in F. Note the presence of both recipient (GFP-/LYVE-1+) and donor (GFP+/LYVE-1+) lymphatics at the distal margin of the wound.
Mentions: In order to investigate the involvement of recipient and donor cells in spontaneous lymphatic regeneration, the tails of athymic nude mice that underwent full-thickness skin excision were repaired with skin grafts harvested from GFP mice. This allowed us to follow the origin of cells by co-localization of GFP and lymphatic markers. Using this technique, the recipient nude mice lymphatics stained positive for lymphatic markers but negative for GFP. The transplanted tissue lymphatics, in contrast, stained positive for both GFP and lymphatic markers (Figure 1). Using fluorescent imaging, we found that high-level GFP expression was maintained in the transplanted skin even 6 weeks after tissue transfer (Figure 1A). The transplanted skin grafts healed quickly and with regrowth of hair and skin appendages by 6 weeks indicating that the grafts had become fully incorporated (Figure 1B). In addition, return of lymphatic function with transport of fluorescence-labeled colloid could be grossly visualized by microlymphangiography as early as 2 weeks after surgery (Figure 1C). This transport was initially driven by interstitial flow (i.e. no discrete lymphatic vessels could be visualized at 2 weeks); however, by 6 weeks honeycomb-like dermal lymphatics were noted in the transferred grafts (primarily at the distal margin) suggesting that the reformed lymphatic vessels are functional (Figure 1D). The lack of complete restoration of the honeycomb-like dermal lymphatic architecture in the mid portion of the graft at this time may reflect a delay in dermal lymphatic regeneration. This concept is supported by previous studies demonstrating that honeycomb-like lymphatic vessels that regenerate in the mouse tail after coverage of the wound with collagen gel are not ordinarily visible until day 60 after surgery and even at this time are only seen at the distal margin of the wound.[21], [22]

Bottom Line: Patterns of VEGF-C expression and macrophage infiltration were temporally and spatially associated with lymphatic regeneration.When compared to mice treated with excision only, there was a 4-fold decrease in tail volumes, 2.5-fold increase in lymphatic transport by lymphoscintigraphy, 40% decrease in dermal thickness, and 54% decrease in scar index in skin-grafted animals, indicating that tissue transfer could bypass damaged lymphatics and promote rapid lymphatic regeneration.This process is temporally and spatially associated with VEGF-C expression and macrophage infiltration.

View Article: PubMed Central - PubMed

Affiliation: The Division of Plastic and Reconstructive Surgery, Department of Surgery, Memorial Sloan-Kettering Cancer Center, New York, New York, United States of America.

ABSTRACT

Introduction: Lymphedema is the chronic swelling of an extremity that occurs commonly after lymph node resection for cancer treatment. Recent studies have demonstrated that transfer of healthy tissues can be used as a means of bypassing damaged lymphatics and ameliorating lymphedema. The purpose of these studies was to investigate the mechanisms that regulate lymphatic regeneration after tissue transfer.

Methods: Nude mice (recipients) underwent 2-mm tail skin excisions that were either left open or repaired with full-thickness skin grafts harvested from donor transgenic mice that expressed green fluorescent protein in all tissues or from LYVE-1 knockout mice. Lymphatic regeneration, expression of VEGF-C, macrophage infiltration, and potential for skin grafting to bypass damaged lymphatics were assessed.

Results: Skin grafts healed rapidly and restored lymphatic flow. Lymphatic regeneration occurred beginning at the peripheral edges of the graft, primarily from ingrowth of new lymphatic vessels originating from the recipient mouse. In addition, donor lymphatic vessels appeared to spontaneously re-anastomose with recipient vessels. Patterns of VEGF-C expression and macrophage infiltration were temporally and spatially associated with lymphatic regeneration. When compared to mice treated with excision only, there was a 4-fold decrease in tail volumes, 2.5-fold increase in lymphatic transport by lymphoscintigraphy, 40% decrease in dermal thickness, and 54% decrease in scar index in skin-grafted animals, indicating that tissue transfer could bypass damaged lymphatics and promote rapid lymphatic regeneration.

Conclusions: Our studies suggest that lymphatic regeneration after tissue transfer occurs by ingrowth of lymphatic vessels and spontaneous re-connection of existing lymphatics. This process is temporally and spatially associated with VEGF-C expression and macrophage infiltration. Finally, tissue transfer can be used to bypass damaged lymphatics and promote rapid lymphatic regeneration.

Show MeSH
Related in: MedlinePlus