The effect of platelet rich plasma on angiogenesis in ischemic flaps in VEGFR2-luc mice
Introduction
Skin flaps are routinely used for reconstruction of large skin defects and deep wounds secondary to trauma, ablative surgery or congenital defects. Ways of improving skin flap survival and the mechanisms involved in the healing process at the recipient site are much-studied matters. The persistence of both partial and complete skin flap necrosis despite the greatest of efforts presents a challenging, multifactorial problem. Adequate vascular supply to the integument is crucial to skin flap survival, especially in comorbid patients. Elevating a skin flap results in varying degrees of hypoxia, which acts as the primary stimulus for vascular changes. The effects of hypoxia on vascular growth and angiogenesis in ischemic tissues have been reported in several studies [1], [2], [3], [4], [5]. Several preconditioning techniques are used to increase ischemic tolerance [6], [7]. Following these procedures, angiogenic agents such as vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF) are increased, leading to new blood vessel formation [8], [9].
A skin flap actually survives as a consequence of flawless wound healing process. Angiogenesis, the process of capillary sprouting from preexisting blood vessels, is essential for wound healing [10], [11]. VEGF, a potent angiogenic, mitogenic and vascular permeability-enhancing protein, is secreted in the cutis by keratinocytes and fibroblasts [5], [12], [13]. VEGF is a pleiotropic factor that acts on a variety of cells, but especially on dermal vascular structures involved in cutaneous angiogenesis. This is because VEGF is one of the growth factors with strong effects on endothelial cells [14], [15]. Activation entails circulating VEGF binding to tyrosine kinase receptors on endothelial cell surfaces; of these, VEGFR2 is the one responsible for regulating angiogenesis [16], [17], [18]. VEGFR2 is expressed in vascular endothelial progenitors [19], and its expression is up-regulated during wound healing [20].
Inadequate angiogenesis leads to loss of tissue function and necrosis of skin flaps. Several studies have examined topical or systemic exogenous VEGF used to improve wound healing processes and reduce flap failure during flap surgery [21], [22], [23]. Platelet-rich plasma (PRP) is an available alternative to exogenous VEGF therapy as a source of growth factors to stimulate angiogenesis. At present, however, no study has used bioluminescence imaging (BLI) to evaluate the effect of PRP on VEGFR2. PRP is a rich source of growth factors, has been found effective in accelerating significant tissue repair and regeneration, and releases massive quantities of platelet growth factors [24], [25]. It is a concentration of autologous platelets in a small volume of plasma containing several growth factors including epidermal growth factor (EGF), fibroblast growth factor (FGF), insulin-like growth factor (IGF), keratinocyte growth factor (KGF), platelet-derived growth factor (PDGF), transforming growth factor-beta (TGF-β) and vascular endothelial growth factor (VEGF) [26], [27]. The secretion of growth factors in PRP starts with platelet activation immediately following the addition of calcium, a necessary cofactor for degranulation of the alpha granules [28]. Recently the use of PRP gel in wound healing has become popular in nearly all fields of surgery, including orthopedic, thoracic and vascular surgery, as well as in dentistry and reconstructive oral maxillofacial surgery [25], [28], [29], [30], [31], [32].
Increased production of VEGF in ischemic tissues such as myocardial ischemia or ischemic retinopathy is well described [2], [3], [4], [15]. Since local hypoxia is an inducer of VEGF activity in wounds, and since PRP improves wound healing, it would be beneficial to study their combined use in flap survival and healing.
In vivo BLI in living transgenic mice has been employed for several years in widely ranging research fields, particularly in cancer research, where it is used to monitor the efficacy of drug treatment on the survival of implanted cancer cells or to analyze tumor metastasis [33], [34]. The technique allows non-invasive longitudinal imaging of markers of gene expression in vivo by luciferase-catalyzed reactions. However, the technique has not yet been used to study flap healing and flap survival.
For the present study, we used BLI to monitor the angiogenesis in a murine axial pattern skin flap experimental model designed to examine the effects of PRP and hypoxia in ischemic tissue during flap elevation and microsurgical anastomosis. Even though the beneficial effects of PRP and hypoxia have been described separately in several previous studies, no conclusive experimental therapeutic evidence is available regarding their combined use. Nor have VEGFR2 analyses been used to study their effects. BLI is a simple, quantifiable and non-invasive technique that enables longitudinal studies on transgenic mice. Whether BLI could be used to determine VEGFR2 expression levels during the flap healing process was unclear. In order to address these questions we performed our experiments on transgenic VEGFR2-luc mice, evaluated VEGFR2 activity and flap viability, and analyzed VEGF concentrations and newly formed blood vessels in tissue sections.
Section snippets
Surgical procedure
All animal experiments were conducted in accordance with the guidelines of the German animal protection statute and approved by the governmental review committee.
FVB/N-Tg(VEGFR2-luc)Xen homogenously gender-matched mice of 10–12 weeks of age were used in this study. The animals were housed in a special, temperature- and humidity-controlled, pathogen-free environment on a cycle of 12 h of light and 12 h of darkness, and were allowed free access to food and water.
After flap elevation, only those
Assessment of VEGFR2 gene activation during flap healing in vivo
In vivo longitudinal measurements on VEGFR2-luc mice were performed using an IVIS Lumina Imaging System 100 Series in order to study transcriptional activation of VEGFR2 and its translation into immunohistological vessel formation. To mimic neovascularisation by flap healing we performed an axial skin island flap on mice (Fig. 1). The main vascular network of the lateral thoracic artery and vein along with their abundant connections could be easily observed on the underface of this flap (Fig. 1
Discussion
Improved angiogenesis is known to facilitate flap healing and survival and thus to reduce flap loss. Local hypoxia in ischemic tissue triggers the hypoxia response element HIF-1 alpha, which results in enhanced production of VEGF [35], [36]. Autologous PRP may be capable of improving wound healing in that it releases growth factors such as VEGF, PDGF, FGF [26], [27], [37]. In this study, we investigated the clinical benefits of PRP and ischemia altered growth factors and genes, for which it has
Conclusions
In the present study our data have revealed that PRP and ischemia together produce synergistic therapeutic stimuli for improved angiogenesis by enhancing VEGFR2 and VEGF expression in a surgical wound. Thus, we can provide local application of PRP on the undersurface of a tissue flap following ischemic period can lead to a significant improvement of flap healing and flap survival levels. In conclusion, these results suggest that using PRP in microsurgical free tissue transfers might reduce the
Disclosures
The authors have no conflicts of interest to disclose.
Acknowledgments
We wish to thank Mr. Wolfgang Graulich for the drawing. Furthermore we thank S. Echterhagen, M. Nicolau, L. Shen and A. Rüben for their excellent technical assistance and Assoc. Prof. Cengizhan Acikel from FAVOR laboratories of Gülhane Military Medical Academy, Ankara. This study was supported in part by the Deutsche Forschungsgemeinschaft (DFG) (DFG No. PU 214/3-2: 4-2; 5-2) and by a grant from the Interdisciplinary Centre for Clinical Research (IZKF) within the faculty of Medicine at the RWTH
References (48)
- et al.
Keratinocyte-derived vascular permeability factor (vascular endothelial growth factor) is a potent mitogen for dermal microvascular endothelial cells
J Invest Dermatol
(1995) - et al.
Migration of human monocytes in response to vascular endothelial growth factor (VEGF) is mediated via the VEGF receptor flt-1
Blood
(1996) - et al.
High affinity VEGF binding and developmental expression suggest Flk-1 as a major regulator of vasculogenesis and angiogenesis
Cell
(1993) - et al.
Expression of vascular endothelial growth factor and vascular endothelial growth factor receptor-2 (KDR/Flk-1) in ischemic skeletal muscle and its regeneration
Am J Pathol
(2002) - et al.
Signal transduction by VEGF receptors in regulation of angiogenesis and lymphangiogenesis
Exp Cell Res
(2006) - et al.
Expression and proteolysis of vascular endothelial growth factor is increased in chronic wounds
J Invest Dermatol
(2000) - et al.
The effects of VEGF on survival of a random flap in the rat: examination of various routes of administration
Br J Plast Surg
(2000) - et al.
Platelet-rich plasma: growth factor enhancement for bone grafts
Oral Surg Oral Med Oral Pathol Oral Radiol Endod
(1998) - et al.
Platelet gel: an autologous alternative to fibrin glue with applications in oral and maxillofacial surgery
J Oral Maxillofac Surg
(1997) - et al.
Monitoring luciferase-labeled cancer cell growth and metastasis in different in vivo models
Cancer Lett
(2005)
Adult vasculogenesis occurs through in situ recruitment, proliferation, and tubulization of circulating bone marrow-derived cells
Blood
Platelet-rich plasma: evidence to support its use
J Oral Maxillofac Surg
A novel murine island skin flap for ischemic preconditioning
J Surg Res
An extended dorsal island skin flap with multiple vascular territories in the rat: a new skin flap model
J Surg Res
Respiratory gas tensions and pH in healing wounds
Am J Surg
Vascular endothelial growth factor (VEGF) and its receptors
FASEB J
Vascular endothelial growth factor induced by hypoxia may mediate hypoxia-initiated angiogenesis
Nature
Vascular delay revisited
Plast Reconstr Surg
The angiogenic peptide vascular endothelial growth factor is expressed in foetal and ruptured tendons
Virchows Arch
The angiogenic peptide vascular endothelial growth factor (VEGF) is expressed in chronic sacral pressure ulcers
J Pathol
Ischemic preconditioning improves the survival of skin and myocutaneous flaps in a rat model
Plast Reconstr Surg
Preconditioning with ischemia: a delay of lethal cell injury in ischemic myocardium
Circulation
Basic fibroblast growth factor expression following surgical delay of rat transverse rectus abdominis myocutaneous flaps
Plast Reconstr Surg
Vascular endothelium growth factor, surgical delay, and skin flap survival
Ann Surg
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These authors contributed equally to this work.