Abstract

   The effectiveness of platelet-rich plasma (PRP) for the treatment of
   Achilles tendon disorders still needs to be evaluated through a series
   of prospective studies, but genomic analysis can reveal the existence
   of complementary PRP treatment options. Based on the 96 platelet
   activation-related genes in the Kyoto Encyclopedia of Genes and Genomes
   (KEGG) database, we performed Gene Ontology functional enrichment
   analysis and KEGG enrichment analysis, pathway correlation analysis,
   and enrichment mapping to determine the enrichment results of the gene
   set enrichment analysis and found that the cAMP signalling pathway may
   be the key to enhancing the effectiveness of PRP treatment. The cAMP
   signalling pathway interacts with the Rap1 signalling pathway and
   cGMP-PKG signalling pathway to mediate the entire pathophysiological
   process of Achilles tendon disease. Moreover, ADCY1-9 may be the key to
   the activation of the cAMP signalling network. Further based on the
   data in the Gene Expression Omnibus database, it was found that ADCY4
   and ADCY7 may be the players that play a major role, associated with
   the STAT4-ADCY4-LAMA5 axis and the GRbeta-ADCY7-SEMA3C axis, which is
   expected to be a complementary target for enhancing the efficacy of PRP
   in the treatment of Achilles tendon disease.

   Keywords: Platelet-rich plasma, Achilles tendon disease, cAMP,
   Adenylate cyclase, Complementary target
     __________________________________________________________________

   Core Tip: The cAMP signalling pathway may be the key to enhancing the
   effectiveness of platelet-rich plasma (PRP) in the treatment of
   Achilles tendon disease. ADCY1-9 may be the key to activating the cAMP
   signalling network and is expected to be a complementary target for
   enhancing the effectiveness of PRP in the treatment of Achilles tendon
   disease.

TO THE EDITOR

   We read with great interest the study by Huang et al[[28]1], which
   reported that platelet-rich plasma (PRP) injections alone had no
   significant efficacy on patients who suffered Achilles tendon rupture
   or Achilles tendinopathy and that PRP alone improved ankle mobility
   after 12 months of treatment. Moreover, Dan’s team suggested that the
   method of PRP preparation and the active ingredient, frequency of
   injection, injection dose, and injection site may be factors
   influencing the uncertainty of clinical outcomes.

   We agree with the authors that the assessment of the effectiveness of
   PRP in the treatment of Achilles tendinopathy needs to be advanced by a
   series of prospective studies, which require blinding, randomization,
   and control of variables. In theory, the pathophysiologic process of
   Achilles tendinopathy is associated with localized inflammation of the
   Achilles tendon, with further degenerative lesions leading to complete
   Achilles tendon rupture[[29]2,[30]3]. PRP, obtained by centrifugation
   of whole blood, allows the inclusion of supraphysiologic concentrations
   of growth factors. The release of these growth factors is effectively
   activated, leading to increased regeneration and healing at the site of
   injection[[31]4,[32]5]. Moreover, PRP also treats tendon tissue by
   inducing type I collagen and glycosaminoglycan deposition[[33]5]. The
   central key to these beneficial biological processes in the treatment
   of Achilles tendon disease is platelet activation, and the inability to
   effectively activate platelets may be one of the reasons why PRP is so
   ineffective in treating this disease. As the field of genomics has
   developed and transformed, it has allowed us to more accurately
   understand the keys to complex biological processes, facilitating the
   development of more precise and effective therapeutic regimens. With
   respect to the relevant genes in the “map04611: Platelet activation”
   ([34]https://www.genome.jp/entry/pathway+map04611) pathway in the Kyoto
   Encyclopedia of Genes and Genomes (KEGG) database, we used
   bioinformatics analysis to provide a theoretical basis for the
   potential activation of platelets in PRP.

   Gene ontology (GO) annotation of 96 genes in the “platelet activation”
   signalling pathways using the “enrichGO” function of the
   “clusterProfiler” R package[[35]6] revealed that, under the
   classification of biological processes, in addition to platelet
   activation-related pathways (Figure [36]1A), the “wound healing”,
   “homotypic cell-cell adhesion” and “cAMP biosynthetic process” pathways
   were significantly enriched. In terms of cellular components, cell
   membrane-related pathways were more enriched, which may be a
   prerequisite for the activation of “cell-substrate junction” pathways.
   In terms of molecular function, the phosphatidylinositol-related
   pathway was activated, which may be a preparation for “scaffold protein
   binding”. Furthermore, using the “pairwise_termsim” function of the
   “enrichplot” R package to calculate the correlation between the
   enriched GO pathways and the enrichment map for the enrichment results
   of the gene set enrichment analysis by the “emapplot” function revealed
   that “platelet activation” was correlated with “wound healing”,
   “vascular process in circulatory system”, “cell-matrix-substrate
   adhesion”, and other biological functions closely related to Achilles
   tendon recovery (Figure [37]1B). In addition, cAMP biosynthesis and
   metabolism appear to constitute another functional subgroup resulting
   from platelet activation. Plotting the Gene-Concept Network using the
   “cnetplot” function revealed that none of the pathways, such as “wound
   healing” and “cAMP biosynthetic process”, had common genes but were
   associated with “regulation of body fluid levels” (Figure [38]1C).
   However, in the KEGG database, all 96 genes were identified as related
   to “platelet activation”. This result suggested that the specific role
   of cAMP-related functions in platelet activation is unclear. The cAMP
   signalling pathway, one of the well-known is the protein kinase A (PKA)
   system, is a type of cyclic nucleotide system that regulates key
   physiological processes such as nodal metabolism, secretion, calcium
   homeostasis, muscle contraction, cell fate and gene
   transcription[[39]7]. These processes are closely related to the local
   inflammatory response and immune response of the Achilles tendon, the
   metabolism and differentiation of fibroblasts, and the senescence and
   apoptosis of fibroblasts[[40]2,[41]8,[42]9]. Overall, these
   physiological conditions are known to characterize Achilles tendon
   inflammation, Achilles tendon recovery, and degenerative Achilles
   tendon disease.

Figure 1.

   [43]Figure 1
   [44]Open in a new tab

   Gene ontology enrichment analysis of 96 platelet activation-associated
   genes. A: Bubble plot of gene ontology (GO) functional enrichment
   analysis; B: Association network between GO terms that were enriched;
   C: Enrichment maps between GO terms and genes.

   The “enrichKEGG” function was used to analyse the KEGG pathways
   associated with these 96 platelet activation-related genes (Figure
   [45]2A), and the Rap1, cGMP-PKG, and cAMP signalling pathways were
   found to be enriched. This further verified the results of the GO
   analysis, suggesting that cAMP-related functions might be involved in
   the alteration of low platelet activation. The enrichment map generated
   by the “emapplot” function shown in Figure [46]2B reveals that platelet
   activation and the signalling pathways of Rap1, cGMP-PKG, and cAMP are
   strongly associated and do not exist in relatively large subgroups of
   the pathways. The Rap1 signalling pathway is involved mainly in the
   regulation of cell adhesion and cell junctions, as well as the
   processes of cell migration, polarization, proliferation, and
   survival[[47]10,[48]11]. In Achilles tendon disease, regulation of the
   Rap1 signalling pathway may involve aspects of fibroblast metabolism,
   differentiation, and repair, thereby affecting Achilles tendon
   development and repair. The second messenger cAMP stimulates Epac1, a
   Rap guanylate exchange factor. cAMP activates Epac1 migration to the
   plasma membrane, activates Epac-Rap1 signalling, and enhances
   integrin-mediated cell adhesion[[49]12,[50]13]. The regulation of the
   cGMP-PKG signalling pathway may involve inflammatory responses, immune
   responses, and apoptosis, thus affecting the pathogenesis and
   pathological processes of diseases such as Achilles tendonitis,
   Achilles tendon rupture, and Achilles tendon degeneration. The
   cross-interaction between ATP and GTP also leads to the interaction of
   cGMP with cAMP, which are also second messengers[[51]14]. Gene-concept
   network analysis revealed that ADCY1-9 were common genes for platelet
   activation, Rap1, cGMP-PKG, cAMP, and cytokine activation (Figure
   [52]2C), which was also consistent with the results of the GO analysis
   (Figure [53]1C). These results suggest that ADCY1-9 may be key to the
   cAMP pathway and platelet activation pathway and are closely related to
   a large functional subgroup of the GO pathway beyond “platelet
   activation”. Interaction network analysis of these 96 platelet-related
   genes using the STRING database ([54]https://string-db.org/) revealed
   that ADCY1-9 may be the upstream genes of AKT1 (Figure [55]3), which
   may be the key to ameliorating the inability of platelets to be
   activated efficiently.

Figure 2.

   [56]Figure 2
   [57]Open in a new tab

   Kyoto Encyclopedia of Genes and Genomes enrichment analysis of 96
   platelet activation-associated genes. A: Bubble plot of the Kyoto
   Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis;
   B: Association network between KEGG pathways that were enriched; C:
   Enrichment maps between KEGG signalling pathways and genes.

Figure 3.

   [58]Figure 3
   [59]Open in a new tab

   Protein-protein interaction network of 96 platelet activation-related
   genes.

   The adenylate cyclase (ADCY) family of genes encodes a
   membrane-associated enzyme that catalyses the formation of
   cAMP[[60]15], which is in complete agreement with the results of GO and
   KEGG enrichment analyses. We can further speculate that if activated,
   ADCY1-9 stimulate the cAMP signalling pathway, which in turn leads to
   the upregulation of the Rap1 and cGMP-PKG signalling pathways,
   ultimately completing the global mobilization of the “platelet
   activation” pathway. ADCY1-9 can be used as an adjunctive target for
   RPR therapy to enhance its effectiveness in treating Achilles tendon
   disorders, such as Achilles tendon rupture, Achilles tendonitis, and
   degenerative Achilles tendon diseases.

   Further, we searched the Gene Expression Omnibus (GEO) database for
   data on healthy human platelets sequenced in the [61]GSE178158
   cohort[[62]16], and 65 samples from the [63]GSE178158 cohort were
   included, with 100 healthy human whole blood samples from the
   [64]GSE134080 cohort used as controls[[65]17]. Comparison of the
   differences in the expression levels of ADCY1-9 in platelets and whole
   blood revealed (Figure [66]4A) that ADCY2, 6, and 8 were highly
   expressed in platelets, while ADCY4, 5, 7, and 9 were lowly expressed
   in platelets, and the rest did not differ. Cartilage tissue sequencing
   data from the GEO database for osteoarthritic diseases were also
   obtained, which included 40 osteoarthritic disease samples and 10
   healthy cartilage tissue samples from the [67]GSE51588 cohort[[68]18],
   and 20 osteoarthritic disease samples and 18 healthy cartilage tissue
   samples from the [69]GSE114007 cohort were also included[[70]19].
   Comparative analysis revealed (Figure [71]4B and [72]4C) that ADCY1, 4,
   and 7 were all highly expressed in osteoarthritic disease samples,
   while ADCY3 was all highly expressed in healthy cartilage tissue
   samples. Correlation analysis of ADCY 1-9 expression levels and
   clustering by the ward.D2 method (Figure [73]4D) showed that the
   expression level of adenylate cyclase was synergistic in both blood
   samples and cartilage tissue samples, which was mainly represented by
   ADCY 4 and ADCY 7. The low expression of ADCY 4 and ADCY 7 in platelets
   is consistent with healthy cartilage tissue, and they may be the
   Achilles heel of osteoarthritic diseases. The promoter sequences of
   ADCY4 (chr14: 24, 318, 359-24, 335, 071) and ADCY7 (chr16: 50, 266,
   551-50, 318, 135) were searched in the UCSC database
   ([74]https://genome.ucsc.edu/), and the 2000 nt region upstream of the
   transcriptional start site was set as the selected promoter region. The
   obtained sequences were entered into the PROMO database
   ([75]https://alggen.lsi.upc.es/cgi-bin/promo_v3/promo/promoinit.cgi?dir
   DB=TF_8.3)[[76]20,[77]21], with species selection of Homo sapiens and
   tolerance set at 5% to retrieve site-bound transcription factors. As
   shown in Figure [78]4E and [79]F, 48 transcription factors were
   retrieved for ADCY4, whereas 56 were retrieved for ADCY7. Differential
   expression analysis in platelets vs whole blood and correlation
   analysis in the osteoarthritic disease cohort revealed (Figure [80]4G)
   that STAT4 is either the upstream transcription factor of ADCY4, while
   the upstream transcription factor of ADCY7 may be GR-beta. By The Human
   Protein Atlas ([81]https://www.proteinatlas.org/) searching which
   proteins could be secreted and analyzing the correlation of all
   secreted proteins (Figure [82]4H)[[83]22], we concluded that ADCY 4 may
   cause increased secretion of LAMA 5, while ADCY 7 caused SEMA3C.
   Surprisingly, as shown in Figure [84]4I, both LAMA 5 and SEMA3C were
   highly expressed in the osteoarthritic disease samples. These results
   prove that the STAT 4-ADCY 4-LAMA 5 axis and the GRbeta-ADCY 7-SEMA3C
   axis may regulate the occurrence in osteoarthritic diseases.

Figure 4.

   [85]Figure 4
   [86]Open in a new tab

   The STAT4-ADCY4-LAMA5 axis and the GRbeta-ADCY7-SEMA3C axis are either
   key to osteoarthritic diseases. A: Differential expression levels of
   adenylate cyclase in platelets and whole blood (data from
   [87]GSE178158, [88]GSE134080 cohorts); B: Differential expression
   levels of adenylate cyclase in platelets and whole blood (data from
   [89]GSE51588 cohort); C: Adenylate cyclase expression level difference
   exists in platelets and whole blood (data from [90]GSE114007 cohort);
   D: There is a very strong correlation between the expression level of
   adenylate cyclase (left: [91]GSE134080 cohort, center: [92]GSE51588
   cohort, right: [93]GSE114007 cohort); E: Can be combined with the ADCY4
   promoter region; F: Transcription factors that can bind to ADCY7
   promoter region; G: There is an extremely strong positive correlation
   between STAT4 and ADCY4 expression levels, and between GR-beta and
   ADCY7 expression levels; H: There is an extremely strong positive
   correlation between LAMA5 and ADCY4 expression levels, and between
   SEMA3C and ADCY7 expression levels; I: Both LAMA5 and SEMA3C were
   highly expressed in osteoarthritic disease samples. ^aP < 0.05, ^bP <
   0.01, ^cP < 0.001, and ^dP < 0.0001.

   Based on the above bioinformatics analysis, we speculate that the role
   of adenylate cyclase in osteoarthritic disease is very important, and
   the poor treatment effect of Achilles tendon disease may be that the
   cAMP signaling pathway is not effectively activated, and the key point
   may lie in ADCY 4 and ADCY 7. Both STAT 4-ADCY 4-LAMA 5 axis and
   GRbeta-ADCY 7-SEMA3C axis are suspected to mediate the occurrence of
   osteoarthritic disease, and the relevant treatment options can be
   explored as a theoretical basis.

Footnotes

   Conflict-of-interest statement: All the authors declare no conflicts of
   interest.

   Provenance and peer review: Unsolicited article; Externally peer
   reviewed.

   Peer-review model: Single blind

   Peer-review started: October 26, 2023

   First decision: December 12, 2023

   Article in press: January 16, 2024

   Specialty type: Orthopedics

   Country/Territory of origin: China

   Peer-review report’s scientific quality classification

   Grade A (Excellent): 0

   Grade B (Very good): 0

   Grade C (Good): C

   Grade D (Fair): 0

   Grade E (Poor): 0

   P-Reviewer: Nezhady MAM, Iran S-Editor: Liu JH L-Editor: A P-Editor:
   Zhao S

Contributor Information

   Jing-Yi Sun, College of Basic Medical Science, Qilu Medical University,
   Zibo 255300, Shandong Province, China.

   Cai Li, College of Basic Medical Science, Qilu Medical University, Zibo
   255300, Shandong Province, China.

   Feng-Ying Du, Department of Gastroenterological Surgery, Shandong
   Provincial Hospital of Shandong First Medical University, Jinan 250021,
   Shandong Province, China. sdudfy@126.com.

References