CYR61 improves muscle force recreation in a rabbit trauma model

Abstract

BACKGROUND:

Critically elevated compartment pressures after complicated tibial fractures may result in fibrosis and therefore scarring of muscles with impaired function. Several studies have shown a relationship between angiogenesis and more effective muscle regeneration. Cysteine-rich angiogenic inducer 61 (CYR61) is associated with angiogenesis but it is not clear whether it would restore muscle force, reduce scarring or improve angiogenesis after acute musculoskeletal trauma.

OBJECTIVE:

We researched whether local application of CYR61 (1) restores muscle force, (2) reduces scar tissue formation, and (3) improves angiogenesis.

METHODS:

We generated acute soft tissue trauma with temporary ischemia and increased compartment pressure in 22 rabbits and shortened the limbs to simulate surgical fracture debridement. In the test group, a CYR61-coated collagen matrix was applied locally around the osteotomy site. After 10 days of limb shortening, gradual distraction of 0.5 mm per 12 hours was performed to restore the original length. Muscle force was measured before trauma and on every fifth day after trauma. Forty days after trauma we euthanized the animals and histologically determined the percentage of connective and muscle tissue. Immunohistology was performed to analyze angiogenesis.

RESULTS:

Recovery of preinjury muscle strength was significantly greater in the CYR61 group (2.8 N; 88%) as compared to the control (1.8 N; 53%) with a moderate reduction of connective tissue (9.9% vs. 8.5%). Immunohistochemical staining showed that blood vessel formation increased significantly (trauma vs. control 38.75 $\pm$ 27.45 mm ${}^{2}$ vs. 24.16 $\pm$ 19.81 mm ${}^{2}$ ).

CONCLUSIONS:

Local application of CYR61 may improve restoration of muscle force and accelerate muscle force recovery by improving angiogenesis and moderately reducing connective tissue.

Keywords

CYR61 skeletal musle regeneration trauma angiogenesis

1. Introduction

Muscle damage after musculoskeletal trauma is a prevalent problem which is often accompanied by life-long functional impairments. Angiogenesis plays a pivotal role in muscle regeneration and bone healing. This process is disturbed in high energy injuries with open fractures of the lower limb associated with soft tissue damage, bone defects, and impaired local perfusion. Initial treatment of this fracture type includes radical soft tissue and bone debridement following stabilization with an external fixator [1]. One of the major possible complications of these injuries is acute compartment syndrome (ACS), which most commonly is treated by fasciotomy. In contrast, injuries with vast bone and soft tissue defects can be acutely shortened of approximately 10% as a salvage procedure to close resulting bone defects and to reduce the intracompartimental pressure [2, 3]. Thus, critically elevated compartment pressure is decreased to physiological levels, and therefore soft tissue perfusion improves.

Normally, various cellular responses are well-orchestrated during the regeneration process. There is a close relationship between maintaining blood supply and muscle regeneration, indicating revascularization plays an essential role in the progress of muscle regeneration [4]. Reversely, impaired perfusion leads to disturbed angiogenesis and with it to a compromised regeneration process. However, formation of a functional vascular network is a complex process that requires several angiogenic factors in order to obtain capillary sprouting as well as to induce growth and remodeling of collateral arteries [5]. Taken together, these observations suggest newly developed capillaries would help to provide the injured area with oxygen and substrates and therefore aid in the regenerative process.

CYR61 (CCN1), first reported to be secreted in fibroblasts, belongs to the CCN family and is a heparin-binding, extracellular matrix-associated protein that mediates endothelial cell adhesion, proliferation, migration and neovascularization by binding to integrin $\alpha$ 2 $\beta$ 3 [6]. This protein is expressed in many types of vascular cells, such as endothelial and smooth muscle cells [7]. It is functionally capable of inducing angiogenic response by activating plasma membrane receptors of endothelial cells, and plays important roles in development and wound healing [8]. Besides, myofibroblast senescence is triggered by CYR61 [9].

Soft tissue trauma not only leads to reduced muscle force attributable to scarring of the muscle fibers, but also to insufficient callus [10]. Because ischemia leads to necrosis of muscle tissue, treatment with proangiogenic factors might be a novel approach in the treatment of damaged muscle after trauma. The purpose of this study was to investigate the influence of locally applied CYR61 on musculoskeletal trauma. Therefore, we investigated the effect of CYR61 on muscle regeneration. We hypothesized that local administration of CYR61 (1) restores muscle force, (2) reduces scar tissue formation, and (3) improves angiogenesis.

2. Methods

Principles of laboratory animal care were followed. The animal protocol was designed to minimize pain or discomfort to the animals. The animals were acclimatized to laboratory conditions (23 ${}^{\circ}$ C, 12 h/12 h light/dark, 50% humidity, ad libitum access to food and water) for 2 weeks prior to experimentation. On day 40 all animals were euthanized (intracardiac injection of 1 ml T61 ${}^{\@setsize{\scriptsize}{9.5pt}{\viiipt}{\@viiipt}\textregistered}$ ; Hoechst GmbH, Munich, Germany) for tissue collection. The study protocol was approved by the Institutional Animal Care and Use Committee and the Government Animal Control.

22 New Zealand White (NZW) rabbits were divided into two equal groups. Both groups underwent the same standardized musculoskeletal trauma to one lower leg resulting in a critical elevated intracompartmental pressure of the tibialis anterior muscle [11]. One group (test) was treated locally with CYR61 whereas in the other group no CYR61 was applied (control). To compare the effect of the treatment on muscle force restoration, muscle force was measured before trauma and every fifth day after trauma until euthanasia. To study the effect of CYR61 on scar tissue formation and regeneration of muscle tissue as well as neoangiogenesis, the specimens were examined histologically, histomorphometrically and immunohistochemically. Animals were administered general anaesthesia for all procedures. The average weight of the animals was 3.8 kg. Data from these same animals was reported seperately to explore the effects of CYR61 on callus [12]. To highlight the effect of CYR61 on two different tissues (muscle and bone) and to emphasize different potential therapeutic strategies, it appears sensible to publish these results separately.

2.1 Musculoskeletal trauma

A tourniquet was placed on the upper thigh to stop arterial blood flow for 90 minutes. During the first 30 minutes of ischemia a contusion clamp (10-mm aluminum base, compression load of 100 kilo Pascal $=$ 10 Newtons/cm ${}^{2}$ ) was placed directly on the tibialis anterior muscle belly 15 mm below its origin. Arterial blood pressure was documented continuously after cannulating the medial auricular artery (PowerLab 4/35, Scope v.4.1.4 for Mac, ADInstruments GmbH, Spechbach, Germany). The compartment pressure was gauged using a piezoelectric transducer (KODIAG; Braun-Dexon GmbH, Spangenberg, Germany). Bilateral pressure measurements confirmed the onset of a critically elevated intracompartmental pressure (defined as pressure greater than 30 mmHg) on the traumatized side. A unilateral external fixator (Orthofix ${}^{\@setsize{\scriptsize}{9.5pt}{\viiipt}{\@viiipt}{\textregistered}}$ M-103; Orthofix SRL, Verona, Italy) was applied to the anteromedial aspect of the tibia [13]. Both groups underwent osteotomy of the tibia with a 10-mm diaphyseal block being removed. Thus, the traumatized leg was shortened. After a latency period of 10 days, original length was restored via gradual distraction (0.5 mm/12 h) using the external fixator.

2.2 CYR61 application

In the test group ( $n=$ 11), the osteotomy was sheathed with a 1 $\times$ 0.5 $\times$ 0.5-mm collagen matrix coated with 25 $\mu$ g CYR61 surrounded by the traumatized tibialis anterior muscle. Hereby, the collagen matrix was contiguous to muscle (outer site) and bone (inner site). In the control group ( $n=$ 11), no collagen matrix was applied.

2.3 Muscle force measurements

Before surgery, we measured the baseline dorsiflexion force for each limb of each animal. These data were used as reference values for normalization. To measure the dorsiflexion force, a bipolar electrode for transcutaneous stimulation (amplitudes of 5.1 mA for durations of 2.56 ms) of the peroneal nerve was used. Each measurement consisted of 20 single fast signals at 50-ms intervals. Measurements were made at an interval of 5 days. On every testing day they were repeated four times on each side (traumatized and nontraumatized legs in each animal), resulting in 80 values per animal per side, which are presented as an overall average value. The contralateral side (nontraumatized) served as the paired control. Bilateral force measurements were obtained at Days 0 (before trauma), 5, 10, 15, 25, and 30 after trauma [11].

2.4 Histomorphometric analysis

On day 40 animals were euthanized (intracardiac injection of T61 ${}^{\@setsize{\scriptsize}{9.5pt}{\viiipt}{\@viiipt}\textregistered}$ ; Hoechst GmbH, Munich, Germany). For further analysis the anterior tibialis muscle was completeley excised. After processing the samples, we embedded the specimens in paraffin. 5 $\mu$ m thick cross sections were prepared at a predefined level in a cross section from the same area of the earlier contusion and were stained histochemically with Heidenhain azan stain for connective and muscle tissue. Light microscopic analysis was used to quantify morphologic changes in muscle and connective tissue in one to five sections from each animal [11]. For determination of the ratio of muscle and connective tissue, single muscle fibers and the area of connective tissue were outlined semiautomatically and counted at 100-fold magnification. We measured digitized images of each muscle (treated and nontreated) using image-editing software (Image-Pro Plus, Version 7.0; Media Cybernetics, Inc, Bethesda, MD, USA; and Cell F, Olympus GmbH, Hamburg, Germany).

2.5 Immunohistochemical analysis

Muscle specimens were stained for blood vessels with $\alpha$ -smooth muscle actin ( $\alpha$ -SMA) antibody (monoclonal mouse antihuman SMA as primary antibody, clone 1A4, 1:50, Dako GmbH, Hamburg, Germany). The second antibody was anti-mouse IgG (horseradish-peroxidase-conjugated anti-mouse IgG, 1:200, Vector Laboratories, Burlingame, CA, USA). Detection and visualization of the specific binding was done with a chromogen-substrate-kit (Liquid DAB-Substrate Pack, Bio Genex Laboratories, San Ramon, CA, USA). Specimens incubated without the primary antibody served as negative controls. For analysis, specimens were inspected with 100-fold magnification. We measured digitized images of each muscle (treated and nontreated) using image-editing software (Cell F, Olympus GmbH, Hamburg, Germany).

2.6 Statistical analyses

Muscle force and muscle ratio (amount of muscle tissue in traumatized leg vs. leg without trauma) were analyzed for differences between control group and test group. Number of vessels (10–50 $\mu$ m) was evaluated for both groups. Normal-propability plots were used to reveal the distribution of the data. Differences between normally distributed groups were analyzed with t-test (paired or unpaired). Non-parametric Wilcoxon test (paired or unpaired) was used for investigating differences between non-normal distributed groups. Descriptive analysis (mean $\pm$ SD, n) and boxplots for each variable and each group were prepared. Scatterplots and Spearman-correlation were used to analyze the relationship between muscle strength and muscle ratio. For statistical analysis we used SPSS 25.0 (SPSS Inc, Chicago, IL, USA) and R 3.5.2 (R Development Core Team [2018]; R: A language and environment for statistical computing; R Foundation for Statistical Computing, Vienna, Austria). The statistical methods of this study were reviewed by Daniela Keller (Dipl. Math.) from Statistik und Beratung, Kürnach, Germany.

3. Results

No animals had soft tissue problems or infections. Two animals suffered from pin loosening wherefore they had to be euthanized. One animal died due to perioperative anaethesiological problems. In all other animals, osseus consolidation occured with correct axes and length in both planes. Compartment pressure measurement at the tibialis anterior muscle showed a pretrauma pressure of 5.7 $\pm$ 1.4 mmHg, an increase to 33.8 $\pm$ 4.0 mmHg posttrauma, and a decrease postsurgery down to pyhsiological levels of 12.4 $\pm$ 2.1 mmHg. After 8, 24 and 48 hours compartment pressure was constant at physiological levels. Postsurgery there was no critical compartment pressure, thus no fasciotomy was necessary. Diastolic blood pressure was measured continously.

There was no difference in force ( $p=$ 0.05) between the treated and the control limbs before trauma. After trauma and surgery, a difference ( $p<$ 0.001) was evident in both groups with the force of all traumatized extremities being less than that of the nontraumatized extremities. There was a significant ( $p=$ 0.0015) difference inbetween the groups in muscle force recreation at the final day of testing. Animals treated with CYR61 regained 88% (2.75 N) of their original strength. Instead, the animals of the control group did not restore more than 53% (1.73 N) (Fig. 1, Table 1) after 30 d posttrauma. Muscle strength continued to improve until the 25th day after surgery. The difference between traumatized and nontraumatized limbs remained even after completion of the distraction in both groups (control group, $p=$ 0.001; CYR61 group, $p=$ 0.12).

Table 1
Absolute values in Newton (N) of the force produced by the dorsiflexion

Days	Mean value (N) (control)	SD	Mean value (N) (CYR61 $+$ )	SD
0	3.16	0.41	3.14	0.11
5	0.19	0.25	0.31	0.18
10	0.35	0.30	0.48	0.13
15	1.00	0.83	1.31	0.47
20	1.43	0.91	2.21	0.28
25	1.73	0.94	2.69	0.41
30	1.73	0.90	2.75	0.50

Figure 1.

Posttraumatic muscle force restoration after 30 days. Animals treated with CYR61 restored 88% (2.8 N) of their original muscle strength in comparison to animals of the control group who restored moderate 53% (1.7 N).

By the final day of testing, more than 50% of the animals in the control group had a force less than 60% of the presurgery baseline, four animals had a measured force greater than 60% of the baseline measure, and just one animal had regained 88% of original muscle strength. As opposed to this, all of the CYR61 treated animals regained more than 80% of the original strength. Three animals regained more than 90% which was not seen in the control group. Compartment pressures of all animals normalized after acute shortening of the limb (Table 2).

Table 2

Muscle force restoration and distribution of connective tissue 30 days after trauma ${}^{*}$

Specimen number	Muscle force	Connective tissue trauma (control-no trauma)	Specimen (CYR61+)	Muscle force (CYR61+)	Connective tissue (CYR61+) (control-no trauma)
1	3.8	3.7 (4.1)	1	83.3	9.3 (2.7)
2	67.0	6.4 (3.2)	2	88.8	8.3 (3.2)
3	85.0	14.5 (2.4)	3	92.6	6.8 (5.6)
4	32.0	14.7 (1.8)	4	84.6	10.9 (4.4)
5	40.0	11.3 (4.0)	5	91.5	7.2 (3.4)
6	51.5	17.4 (2.3)	6	87.3	7.7 (6.1)
7	23.0	15.8 (2.6)	7	92.4	6.6 (3.7)
8	60.0	6.0 (1.7)	8	81.7	11.3 (2.7)
9	78.1	6.5 (1.4)	–
10	87.5	9.0 (2.4)	–
11	57.6	3.3 (1.6)	–
Average	53.2	9.9 (2.5)	Average	87.8	8.5 (4.0)

${}^{*}$ Muscle force and connective tissue values in percent; the pretrauma values are set as baseline (100%); CYR61 $=$ cysteine-rich angiogenic inducer 61.

Histomorphometric evaluation showed 4.5% more connective tissue on the traumatized CYR61 side than compared with the nontraumatized (4% $\pm$ 1.3% versus 8.5% $\pm$ 1.8%). Instead, animals of the control group developed 7.4% more connective tissue in the traumatized leg (9.9% $\pm$ 5.4 vs. 2.5% $\pm$ 0.9). Just two of the CYR61 treated animals developed more than 10% of connective tissue compared to five in the control group. On average, the amount of connective tissue was 1.4% lower in the traumatized legs of the CYR61 treated group (control group: 9.9% ( $\pm$ 5.4) vs. test group: 8.5% ( $\pm$ 1.83); $p=$ 0.9) (Fig. 2). Interestingly, on sides without surgery, connective tissue percentages were significantly higher ( $p=$ 0.009) in the CYR61 control (4% $\pm$ 1.3% for CYR61 compared with 2.5% $\pm$ 0.9% for control) (Table 2). Considering this, a difference overall amount of connective tissue of even 2.9%. A strong positive correlation between muscle force restoration and muscle tissue regeneration was observed in the CYR61 group (Rho $=$ 0.86, $p=$ 0.01).

Figure 2.

Morphological features of muscle and collagen expression (Stain, Heidenhain azan). The muscle fibers are stained red and the connective tissue blue. A: normal muscle; B: uninjured muscle; C $+$ D: traumatized tibialis anterior muscle (control); E $+$ F: traumatized muscle regenerated in the presence of CYR61.

Immunohistochemical staining showed that blood vessel formation increased with a significantly higher number of vessels with crossectional area of 10–50 $\mu$ m in the traumatized leg of the CYR61 group (38.75 $\pm$ 27.45 mm ${}^{2}$ ) compared to the controlateral non-traumatized leg 9.13 $\pm$ 6.08 mm ${}^{2}$ ( $p=$ 0.016) (Fig. 3). In addition, there was a significant higher number of blood vessels in the traumatized leg of the CYR61 group (38.75 $\pm$ 27.45 mm ${}^{2}$ ) than compared to the traumatized legs of the control (24.16 $\pm$ 19.81 mm ${}^{2}$ ) ( $p=$ 0.01) (Fig. 3). Although not all blood vessels contained smooth muscle cells or pericytes and not all pericytes stained positive for $\alpha$ -SMA, our experience with other staining techniques than for angiogenesis (Desmin, alpha-sarcomeric and und Myf-5) showed that immunolabeling with human $\alpha$ -SMA was the most robust method.

Figure 3.

Immunohistochemical staining with $\alpha$ -SMA. Significant increase of vessels in a CYR61 treated traumatized leg (non traumatized tibialis anterior muscle (A, B), traumatized tibialis anterior muscle (control group) (C, D) and traumatized tibialis anterior muscle treated with CYR61 (E, F).

4. Discussion

Open lower limb fractures caused by high energy injuries are often associated with devastating soft tissue damage, bone defects and impaired local perfusion. The natural regeneration process is disturbed. Primary treatment includes radical soft tissue and bone debridement, acute shortening to close bone defects and stabilization with an external fixator [2]. This process is a complex mechanistic cascade composed of the interplay of inflammation, neoangiogenesis, bone remodelling, but also muscle tissue regeneration and scarring of the muscle which leads to functional impairments. Simulating the clinical situation we mimicked a musculoskeletal injury which leads to reduced bone quality and non-reversible muscle damage as shown before [10]. As a consequence of this trauma, the natural coupling between angiogenesis and muscle regeneration, which is a basic prerequisite for the whole regeneration process is diminished. Hypoxia and necrosis of the soft tissues lead to scarring of the muscle with a vast functional deficit. As an angiogenic inducer CYR61 may have a regnerative capacity. However, until now there are no comparable studies showing the effect of CYR61 after acute soft tissue trauma. Therefore, we asked whether local application of CYR61 (1) restores muscle force, (2) reduces scar tissue formation, and (3) improves angiogenesis.

The results of the muscle force measurement showed a significant difference in muscle force restoration inbetween the two groups. The CYR61 treated animals showed a significant increase in muscle force restoration (CYR61: 88% (2.75 N) vs. control: 53% (1.73 N)). The soft tissue trauma might have triggered the response of CYR61 which is classified as an immediate early gene, rapidly induced in response to externally applied mechanical strain in cardiac and smooth muscle cells and in fibroblasts [14, 15]. There is an increased expression of CYR61 in skeletal muscle after eccentric exercise [16, 17]. Fast muscle fibers express more CYR61 compared with slow fibers. The potential function of CYR61 in healthy skeletal muscle after exercise include the induction of angiogenic responses. CYR61 staining in human skeletal muscle was found in ECM surrounding the muscle fibers where blood vessels are also located [17]. As reported before, gene transfer of CYR61 is effective in improving perfusion in ischemic hindlimb model [18]. These observations are in line with our findings that CYR61 has a myogenic potential. The signaling pathways mediated by CYR61 are being intensively studied at the moment, but their role in skeletal muscle regeneration and force restoration is yet to be fully defined.

In animals treated with CYR61, scar tissue formation was reduced moderately which is associated with a higher amount of muscle tissue, indicating CYR61 had a slight antifibrotic and myogenic capacity. Before it was described that myofibroblast senescence is triggered by CYR61. It was shown show that purified CYR61 protein can directly induce fibroblast senescence [9]. Mechanistically, CYR61 induces fibroblast senescence through its direct binding to integrin $\alpha_{6}\beta_{1}$ and cell surface heparan sulfate proteoglycans [19]. Interestingly, after myocardial infarction in mice, CYR61 expression was strongly induced in ischemic myocardium [20]. Furthermore, improvement of the muscular deficit seems to be related to the stimulation of myogenesis and the antiapoptotic and antifibrotic effects of CYR61 in different cells types [8] what might be one approach to explain our results. Another reasonable answer might be that a vigorous inflammatory response is coupled to the rapid synthesis and deposition of extracellular matrix (ECM) which is activated in tissue injuries. When excessive, the resulting fibrosis, scarring and loss of tissue function may lead to deleterious consequences [9]. In adulthood the expression of CYR61 is associated with inflammation, wound healing and injury repair [21, 22, 23].

Here, we demonstrate a moderate antifibrotic effect of CYR61 which was not significant but apparent and which confirms the findings in the literature.

There was an increased amount of vessels in the CYR61 treated limb compared to the contralateral side and to the control group. The potent angiogenic activity of CYR61 is well documented. Among others it was confirmed in a rabbit [18], rat [24] and mouse [25] ischemic hindlimb model. Revascularization may be stimulated by acting as an angiogenic inducer of endothelial cells, as VEGF does, and in addition by acting as chemotactic, mitogenic, and matrix-remodeling factor in fibroblasts [6, 26]. The angiogenic capacity can be attributed to its binding to integrin $\alpha_{v}\beta_{3}$ , a major integrin which is expressed and which has been shown to be in endothelial cells responsible for mediating CYR61 function in promoting endothelial cell adhesion, migration, proliferation, survival, and tubule formation [27]. Besides, it appears closely related to bFGF-mediated angiogenesis [28], whereas bFGF is a major inducer of the CYR61 gene which has been presented as the main angiogenic factor implicated in arteriogenesis [29, 30]. Furthermore, it stimulates the integrin-dependent recruitment of CD34 ${}^{+}$ progenitor cells, thereby enhancing endothelial proliferation and neovascularization [7]. Moreover, even if CYR61 is not mitogenic by itself, it may synergize with growth factors such as bFGF to enhance mitogenesis of endothelial cells [6, 26]. The effects of CYR61 include upregulation of VEGF [31, 32, 33]. Both are transcriptionally induced under hypoxia through the action of hypoxia-inducible factor 1 $\alpha$ (HIF-1 $\alpha$ ). The critical role of CYR61 in vascular injury repair is discussed as inhibition of CYR61 might potentially prevent restenosis after vascular interventions [34]. Recent studies suggested that CCN1 has a central role in angiogenic function during tissue repair by promoting vascular smooth muscle cells proliferation and migration. Besides, it is involved in endothelial cell dysfunction and regulates TNF- $\alpha$ induced endothelial cells apoptosis [35]. Our results are in accordance with the present studies suggesting that CYR61 improves angiogenesis after acute soft tissue trauma.

We recognize limitations to our study. Experimental studies like ours generally must be transferred to human clinical practice with caution. There are no clinical data on the use of CYR61 in human musculoskeletal trauma. We showed a positive effect of CYR61 in rabbit limbs but it needs to be emphasized that these tissues are not identical to human limbs. The inherent differences in anatomy in the rabbit compartment to human patients meant that the dynamics related to an injury with critically elevated compartment pressure induced in this model vary considerably and translation of clinical parameters between models should be carefully examined. However, this problem is common to the majority of animals used as models for compartment syndromes, and does not negate the importance or potential translation of such research.

In conclusion, in the present study, we showed that animals treated with CYR61 after musculoskeletal trauma had a distinct increase in muscle force restoration and the quantity of vessels but there was no significant reduction of fibrotic tissue. These results suggest that CYR61 might promote muscle force recreation by improving angiogenesis. Thus, CYR61 used locally could be an perspectival effective therapeutic option after ischemic traumatic muscular deficit to improve angiogenesis and muscle force.

Footnotes

Acknowledgments

We thank James Ridgley (MD) for critically reviewing the manuscript as a native speaker, and Daniela Keller (Dipl. Math. – statistics) for her excellent support.

Conflict of interest

All ICMJE Conflict of Interest Forms are on file with the publication and can be viewed on request. There are no conflicting interests that are related to the work submitted for consideration of publication.

References

Meffert

Jansen

Frey

SNP

Raschke

Langer

. The Influence of Soft Tissue Trauma on Bone Regeneration after Acute Limb Shortening. Clinical Orthopaedics and Related Research. 2007 Mar; PAP.

Möllenhoff

Josten

Muhr

. [Callotaxis–osteogenesis by stretching – a conservative possibility for restoring leg length after post-traumatic primary tibial shortening? Zentralbl Chir. 1997; 122(11): 970-3.

Meffert

Inoue

Tis

Brug

Chao

. Distraction osteogenesis after acute limb-shortening for segmental tibial defects. Comparison of a monofocal and a bifocal technique in rabbits. J Bone Joint Surg Am. 2000 Jun; 82(6): 799-808.

Brunelli

Rovere-Querini

. The immune system and the repair of skeletal muscle. Pharmacol Res. 2008 Aug; 58(2): 117-21.

Grundmann

Piek

Pasterkamp

Hoefer

. Arteriogenesis: basic mechanisms and therapeutic stimulation. Eur J Clin Invest. John Wiley & Sons, Ltd (10.1111); 2007 Oct; 37(10): 755-66.

Babic

Kireeva

Kolesnikova

Lau

. CYR61, a product of a growth factor-inducible immediate early gene, promotes angiogenesis and tumor growth. Proc Natl Acad Sci USA. National Academy of Sciences. 1998 May 26; 95(11): 6355-60.

Grote

Salguero

Ballmaier

Dangers

Drexler

Schieffer

. The angiogenic factor CCN1 promotes adhesion and migration of circulating CD34+ progenitor cells: potential role in angiogenesis and endothelial regeneration. Blood. American Society of Hematology. 2007 Aug 1; 110(3): 877-85.

Chen

X-Y

. Functional properties and intracellular signaling of CCN1/Cyr61. J Cell Biochem. 2007; 100(6): 1337-45.

Jun

J-I

Lau

. Cellular senescence controls fibrosis in wound healing. Aging (Albany NY). 2010 Sep; 2(9): 627-31.

10.

Meffert

Jansen

Frey

Raschke

Langer

. The influence of soft tissue trauma on bone regeneration after acute limb shortening. Clinical Orthopaedics and Related Research. 2007 Jul; 460: 202-9.

11.

Meffert

Frey

Jansen

Ochman

Raschke

Langer

. Muscle strength quantification in small animals: a new transcutaneous technique in rabbits. J Orthop Res. Wiley Subscription Services, Inc., A Wiley Company. 2008 Nov; 26(11): 1526-32.

12.

Frey

Doht

Eden

Dannigkeit

Schuetze

Meffert

, et al. Cysteine-rich matricellular protein improves callus regenerate in a rabbit trauma model. International Orthopaedics. 2012 Nov; 36(11): 2387-93.

13.

Meffert

Tis

Lounici

Rogers

Inoue

Chao

. Comparison of two systems for tibial external fixation in rabbits. Lab Anim Sci. 1999 Dec; 49(6): 650-4.

14.

Schild

Trueb

. Three members of the connective tissue growth factor family CCN are differentially regulated by mechanical stress. Biochimica et Biophysica Acta (BBA) – Molecular Cell Research. 2004 Apr; 1691(1): 33-40.

15.

Zhou

Herrick

Rosenbloom

Chaqour

. Cyr61 mediates the expression of VEGF, αv-integrin, and α-actin genes through cytoskeletally based mechanotransduction mechanisms in bladder smooth muscle cells. J Appl Physiol. American Physiological Society. 2005 Jun; 98(6): 2344-54.

16.

Chen

Y-W

Hubal

Hoffman

Thompson

Clarkson

. Molecular responses of human muscle to eccentric exercise. J Appl Physiol. American Physiological Society; 2003 Dec; 95(6): 2485-94.

17.

Kivelä

Kyröläinen

Selänne

Komi

Kainulainen

Vihko

. A single bout of exercise with high mechanical loading induces the expression of Cyr61/CCN1 and CTGF/CCN2 in human skeletal muscle. J Appl Physiol. 2007 Oct; 103(4): 1395-401.

18.

Fataccioli

Abergel

Wingertsmann

Neuville

Spitz

Adnot

, et al. Stimulation of angiogenesis by Cyr61 gene: a new therapeutic candidate. Hum Gene Ther. 2002 Aug 10; 13(12): 1461-70.

19.

Campisi

d’Adda di Fagagna

. Cellular senescence: when bad things happen to good cells. Nat Rev Mol Cell Biol. Nature Publishing Group. 2007 Sep; 8(9): 729-40.

20.

Hilfiker-Kleiner

Kaminski

Kaminska

Fuchs

Klein

Podewski

, et al. Regulation of proangiogenic factor CCN1 in cardiac muscle: impact of ischemia, pressure overload, and neurohumoral activation. Circulation. Lippincott Williams & Wilkins; 2004 May 11; 109(18): 2227-33.

21.

Kim

K-H

Won

Cheng

Lau

. The matricellular protein CCN1 in tissue injury repair. J Cell Commun Signal. Springer Netherlands; 2018 Mar; 12(1): 273-9.

22.

Borkham-Kamphorst

Schaffrath

Van de Leur

Haas

Tihaa

Meurer

, et al. The anti-fibrotic effects of CCN1/CYR61 in primary portal myofibroblasts are mediated through induction of reactive oxygen species resulting in cellular senescence, apoptosis and attenuated TGF-β signaling. Biochim Biophys Acta. 2014 May; 1843(5): 902-14.

23.

Lau

. CCN1 and CCN2: blood brothers in angiogenic action. J Cell Commun Signal. Springer Netherlands. 2012 Aug; 6(3): 121-3.

24.

Yin

Liang

Guo

Zhou

Pan

. CCN1 enhances angiogenic potency of bone marrow transplantation in a rat model of hindlimb ischemia. Mol Biol Rep. Springer Netherlands. 2014 Sep; 41(9): 5813-8.

25.

Lebas

Galley

Renaud-Gabardos

Pujol

Lenfant

Garmy-Susini

, et al. Therapeutic Benefits and Adverse Effects of Combined Proangiogenic Gene Therapy in Mouse Critical Leg Ischemia. Ann Vasc Surg. 2017 Apr; 40: 252-61.

26.

Kireeva

Yang

Lau

. Cyr61, a product of a growth factor-inducible immediate-early gene, promotes cell proliferation, migration, and adhesion. Mol Cell Biol. American Society for Microbiology (ASM). 1996 Apr; 16(4): 1326-34.

27.

Leu

S-J

Lam

SC-T

Lau

. Pro-angiogenic activities of CYR61 (CCN1) mediated through integrins αvβ3 and α6β1 in human umbilical vein endothelial cells. J Biol Chem. American Society for Biochemistry and Molecular Biology. 2002 Nov 29; 277(48): 46248-55.

28.

Friedlander

Brooks

Shaffer

Kincaid

Varner

Cheresh

. Definition of two angiogenic pathways by distinct alpha v integrins. Science. 1995 Dec 1; 270(5241): 1500-2.

29.

Arras

Ito

Scholz

Winkler

Schaper

. sMonocyte activation in angiogenesis and collateral growth in the rabbit hindlimb. J Clin Invest. American Society for Clinical Investigation. 1998 Jan 1; 101(1): 40-50.

30.

Rayssac

Neveu

Pucelle

Van den Berghe

Prado-Lourenco

Arnal

J-F

, et al. IRES-based vector coexpressing FGF2 and Cyr61 provides synergistic and safe therapeutics of lower limb ischemia. Mol Ther. 2009 Dec; 17(12): 2010-9.

31.

Chen

Lau

. The angiogenic factors Cyr61 and connective tissue growth factor induce adhesive signaling in primary human skin fibroblasts. J Biol Chem. American Society for Biochemistry and Molecular Biology. 2001 Mar 30; 276(13): 10443-52.

32.

Ivkovic

Yoon

Popoff

Safadi

Libuda

Stephenson

, et al. Connective tissue growth factor coordinates chondrogenesis and angiogenesis during skeletal development. Development. NIH Public Access. 2003 Jun; 130(12): 2779-91.

33.

Dean

Butler

Hamma-Kourbali

Delbé

Brigstock

Courty

, et al. Identification of candidate angiogenic inhibitors processed by matrix metalloproteinase 2 (MMP-2) in cell-based proteomic screens: disruption of vascular endothelial growth factor (VEGF)/heparin affin regulatory peptide (pleiotrophin) and VEGF/Connective tissue growth factor angiogenic inhibitory complexes by MMP-2 proteolysis. Mol Cell Biol. American Society for Microbiology Journals. 2007 Dec; 27(24): 8454-65.

34.

Matsumae

Yoshida

Ono

Togi

Inoue

Furukawa

, et al. CCN1 knockdown suppresses neointimal hyperplasia in a rat artery balloon injury model. Arterioscler Thromb Vasc Biol. Lippincott Williams & Wilkins. 2008 Jun; 28(6): 1077-83.

35.

Zhang

Dai

. The matricellular protein CCN1 regulates TNF-α induced vascular endothelial cell apoptosis. Cell Biol Int. John Wiley & Sons, Ltd. 2016 Jan; 40(1): 1-6.

CYR61 improves muscle force recreation in a rabbit trauma model

Abstract

BACKGROUND:

OBJECTIVE:

METHODS:

RESULTS:

CONCLUSIONS:

Keywords

1. Introduction

2. Methods

2.1 Musculoskeletal trauma

2.2 CYR61 application

2.3 Muscle force measurements

2.4 Histomorphometric analysis

2.5 Immunohistochemical analysis

2.6 Statistical analyses

3. Results

Table 1 Absolute values in Newton (N) of the force produced by the dorsiflexion

Footnotes

Acknowledgments

Conflict of interest

References

Table 1
Absolute values in Newton (N) of the force produced by the dorsiflexion