CXCL12 Isoforms Research Articles

Quantification of increased biologically active CXCL12α plasma concentrations after ACKR3 antagonist treatment in humans

AbstractCXCL12 acts as a chemoattractant by binding to the receptor CXCR4. The (atypical) chemokine receptor ACKR3 (CXCR7) scavenges CXCL12. Antagonism of ACKR3 thus leads to an increase in CXCL12 concentrations that has been used as a pharmacodynamic biomarker in healthy adults. Increased CXCL12 concentrations have also been linked to repair mechanisms in human diseases and mouse models. To date, CXCL12 concentrations have typically been quantified using antibody‐based assays with overlapping or unclear specificity for the various CXCL12 isoforms (α, β, and γ) and proteoforms. Only the N‐terminal full‐length CXCL12 proteoform is biologically active and can engage CXCR4 and ACKR3, but this proteoform could so far not be quantified in healthy adults. Here, we describe a new and fit‐for‐purpose validated immunoaffinity mass spectrometry (IA‐MS) assay for specific measurement of five CXCL12α proteoforms in human plasma, including the biologically active CXCL12α proteoform. This biomarker assay was used in a phase I clinical study with the ACKR3 antagonist ACT‐1004‐1239. In placebo‐treated healthy adults, 1.0 nM total CXCL12α and 0.1 nM biologically active CXCL12α was quantified. The concentrations of both proteoforms increased up to two‐fold in healthy adults compared to placebo following drug administration. At all dose levels, 10% of the CXCL12α was the biologically active proteoform and the simultaneous increase of all proteoforms suggests that a new steady state has been reached 24 h following dosing. Hence, this IA‐MS biomarker assay can be used to specifically measure active CXCL12 proteoform concentrations in clinical trials to demonstrate target engagement and correlate with clinical outcomes.

Microarray analysis to explore the effect of CXCL12 isoforms in a pancreatic pre-tumor cell model.

CXCL12 expression was significantly lower in tumor samples than in corresponding normal samples. CXCL12 expression was significantly positively related to the infiltration levels of T cells, dendritic cells (DCs), immature DCs, cytotoxic cells, Tfh cells, mast cells, B cells, Th1 cells, natural killer (NK) cells, pDCs, neutrophils, and T helper cells (Spearman correlation coefficient > 0.5, P < 0.001) and negatively correlated with the infiltration level of NK CD56bright cells. In addition, pancreatic hTERT-HPNE cells treated with three diverse CXCL12 isoforms exhibited changes mainly in the regulation of the epithelial-mesenchymal transition activation pathway.

Effects of CXCL12 isoforms in a pancreatic pre-tumour cellular model: Microarray analysis.

BACKGROUNDPancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of death among cancers, it is characterized by poor prognosis and strong chemoresistance. In the PDAC microenvironment, stromal cells release different extracellular components, including CXCL12. The CXCL12 is a chemokine promoting the communication between tumour and stromal cells. Six different splicing isoforms of CXCL12 are known (α, β, γ, δ, ε, θ) but their role in PDAC has not yet been characterized.AIMTo investigate the specific role of α, β, and γ CXCL12 isoforms in PDAC onset.METHODSWe used hTERT-HPNE E6/E7/KRasG12D (Human Pancreatic Nestin-Expressing) cell line as a pancreatic pre-tumour model and exposed it to the α, β, and γ CXCL12 isoforms. The altered expression profiles were assessed by microarray analyses and confirmed by Real-Time polymerase chain reaction. The functional enrichment analyses have been performed by Enrichr tool to highlight Gene Ontology enriched terms. In addition, wound healing assays have been carried out to assess the phenotypic changes, in terms of migration ability, induced by the α, β, and γ CXCL12 isoforms.RESULTSMicroarray analysis of hTERT-HPNE cells treated with the three different CXCL12 isoforms highlighted that the expression of only a few genes was altered. Moreover, the α and β isoforms showed an alteration in expression of different genes, whereas γ isoform affected the expression of genes also common with α and β isoforms. The β isoform altered the expression of genes mainly involved in cell cycle regulation. In addition, all isoforms affected the expression of genes associated to cell migration, adhesion and cytoskeleton. In vitro cell migration assay confirmed that CXCL12 enhanced the migration ability of hTERT-HPNE cells. Among the CXCL12 splicing isoforms, the γ isoform showed higher induction of migration than α and β isoforms.CONCLUSIONOur data suggests an involvement and different roles of CXCL12 isoforms in PDAC onset. However, more investigations are needed to confirm these preliminary observations.

Distinguishing Specific CXCL12 Isoforms on Their Angiogenesis and Vascular Permeability Promoting Properties.

Angiogenesis is associated with increased vessel sprouting and permeability. Important mediators of these angiogenic responses include local environment of signaling molecules and supporting extracellular matrix (ECM). However, dissecting the interplay of these instructive signals in vivo with multiple cells and extracellular molecules remains a central challenge. Here, microfluidic biomimicry is integrated with 3D ECM hydrogels that are well-characterized for molecular-binding and mechanical properties to reconstitute vessel-like analogues in vitro. This study focuses on three distinct isoforms of the pro-metastatic chemokine CXCL12. In collagen-only hydrogel, CXCL12-α is the most potent isoform in promoting sprouting and permeability, followed by CXCL12-β and CXCL12-γ. Strikingly, addition of hyaluronan (HA), a large and negatively charged glycosaminoglycan, with collagen matrices selectively increases vessel sprouting and permeability conferred by CXCL12-γ. This outcome is supported by the measured binding affinities to collagen/HA ECM, suggesting that negatively charged HA increases the binding of CXCL12-γ to augment its angiogenic potency. Moreover, it is shown that addition of HA to collagen matrices on its own decreases vessel sprouting and permeability, and these responses are nullified by blocking the HA receptor CD44. Collectively, these results demonstrate that differences in binding to extracellular HA help underlie CXCL12 isoform-specific responses toward directing angiogenesis.

P6292Role of CXCL12gamma isoform and its interactions with heparan-sulfates in post-ischemic cardiac remodeling

Abstract Introduction Myocardial infarction (MI) is a severe ischemic disease precipitating long-term adverse remodeling and heart failure. The chemokine CXCL12/SDF-1 is essential for cardiovascular system development and plays a prominent role in physio-pathological processes such as inflammation, angiogenesis and tissue fibrosis. In addition to the binding to its cognate receptors CXCR4 and CXCR7, CXCL12 interacts with heparan-sulfates (HS) which coordinate its biological activity. We have previously highlighted the essential role of CXCL12/HS interactions in vascular growth and remodeling in the setting of critical limb ischemia. In addition, studies in experimental model of MI revealed a protective role for the CXCL12α isoform, through the regulation of cardiomyocyte survival and recruitment of inflammatory cells. However, in mice, three CXCL12 isoforms (α, β and γ) have been identified and, among them, the CXCL12γ isoform shows an unchallenged ability to cooperate with HS, suggesting a putative pivotal role in tissue repair. Objectives The aim of the study is to analyze the role of CXCL12γ isoform and the importance of CXCL12/HS interactions in post-ischemic cardiac remodeling in an acute model of MI. Methods MI was induced by permanent ligation of the left ascending coronary artery in mice carrying a Cxcl12 gene mutation that precludes interactions with HS (Cxcl12Gagtm) and in Cxcl12γ knock-in animals (Cxcl12γ-KI) harboring CXCL12γ deficiency. Alternatively, the impact of CXCL12γ overexpression and the importance of its interactions with HS was also evaluated in wild-type (WT) mice receiving transcutaneous echo-guided injections of adenovirus encoding WT Cxcl12γ or HS-binding-disabled Cxcl12γ in cardiac tissue. Cardiac function and remodeling have been assessed through echocardiography analysis, evaluation of infarct size, interstitial fibrosis, vascular growth (capillary and arteriole densities) and inflammatory cell infiltration into the cardiac tissue. Results After MI, Cxcl12Gagtm and Cxcl12γ-KI animals exhibit reduction in cardiac function and adverse left ventricular remodeling when compared to their respective WT littermates. Interestingly, overexpression of CXCL12γ in WT mice cardiac restored cardiac function by reducing the size of the infarcted area, interstitial fibrosis and promoting vascular growth. In sharp contrast, HS–binding disabled CXCL12gamma mutants failed to improve cardiac function and to abrogate adverse left ventricular remodeling. Conclusion We show that CXCL12γ isoform plays an important role in the regulation of post-ischemic cardiac function and remodeling and that its interactions with HS are essential for adequate cardiac repair in the setting of acute MI.

CXCL12 and Its Isoforms: Different Roles in Pancreatic Cancer?

CXCL12 is a chemokine that acts through CXCR4 and ACKR3 receptors and plays a physiological role in embryogenesis and haematopoiesis. It has an important role also in tumor development, since it is released by stromal cells of tumor microenvironment and alters the behavior of cancer cells. Many studies investigated the roles of CXCL12 in order to understand if it has an anti- or protumor role. In particular, it seems to promote tumor invasion, proliferation, angiogenesis, epithelial to mesenchymal transition (EMT), and metastasis in pancreatic cancer. Nevertheless, some evidence shows opposite functions; therefore research on CXCL12 is still ongoing. These discrepancies could be due to the presence of at least six CXCL12 splicing isoforms, each with different roles. Interestingly, three out of six variants have the highest levels of expression in the pancreas. Here, we report the current knowledge about the functions of this chemokine and then focus on pancreatic cancer. Moreover, we discuss the methods applied in recent studies in order to understand if they took into account the existence of the CXCL12 isoforms.

CXCL12γ isoform inhibits adverse left ventricular remodeling after acute myocardial infarction

The chemokine CXCL12 is essential for cardiovascular system development and plays a major role in physio-pathological processes such as inflammation, angiogenesis but also tissue remodeling after an ischemic insult. In addition to the binding to its receptors, CXCR4 and CXCR7, CXCL12 interaction with heparan-sulfates (HS) commands its biological activity. Studies in experimental models of myocardial infarction (MI) revealed a protective role for CXCL12α. However, in mice, three CXCL12 isoforms (α, β and γ) have been identified and the CXCL12γ isoform shows an unchallenged ability to cooperate with HS, suggesting a pivotal role in tissue repair. The aim of the study is to analyze the role of CXCL12γ isoform and the importance of CXCL12/HS interactions in post-ischemic cardiac remodeling in an acute model of MI. MI are induced in mice carrying a Cxcl12 gene mutation that precludes interactions with HS (Cxcl12Gagtm) and in Cxcl12γ knock-in animals (Cxcl12γ-KI), harboring CXCL12γ deficiency. Alternatively, the impact of CXCL12γ overexpression is also evaluated in wild-type (WT) mice receiving transcutaneous echo-guided injections of adenovirus encoding Cxcl12γ in cardiac tissue. Cardiac function and remodeling have been assessed through echocardiography analysis and evaluation of infarct size, interstitial fibrosis, capillary and arteriole densities and as well as inflammatory cell infiltration. After MI, Cxcl12Gagtm and Cxcl12γ-KI animals exhibit reduction in cardiac function and adverse left ventricular remodeling when compared to their respective WT littermates. Interestingly, overexpression of CXCL12γ in WT mice normalizes cardiac function by reducing the size of the infarcted area, interstitial fibrosis and promoting vascular growth. We show that CXCL12γ isoform plays an important role in the regulation of cardiac function after MI and that its interactions with HS seems essential for adequate cardiac repair.

Cell, isoform, and environment factors shape gradients and modulate chemotaxis.

Chemokine gradient formation requires multiple processes that include ligand secretion and diffusion, receptor binding and internalization, and immobilization of ligand to surfaces. To understand how these events dynamically shape gradients and influence ensuing cell chemotaxis, we built a multi-scale hybrid agent-based model linking gradient formation, cell responses, and receptor-level information. The CXCL12/CXCR4/CXCR7 signaling axis is highly implicated in metastasis of many cancers. We model CXCL12 gradient formation as it is impacted by CXCR4 and CXCR7, with particular focus on the three most highly expressed isoforms of CXCL12. We trained and validated our model using data from an in vitro microfluidic source-sink device. Our simulations demonstrate how isoform differences on the molecular level affect gradient formation and cell responses. We determine that ligand properties specific to CXCL12 isoforms (binding to the migration surface and to CXCR4) significantly impact migration and explain differences in in vitro chemotaxis data. We extend our model to analyze CXCL12 gradient formation in a tumor environment and find that short distance, steep gradients characteristic of the CXCL12-γ isoform are effective at driving chemotaxis. We highlight the importance of CXCL12-γ in cancer cell migration: its high effective affinity for both extracellular surface sites and CXCR4 strongly promote CXCR4+ cell migration. CXCL12-γ is also more difficult to inhibit, and we predict that co-inhibition of CXCR4 and CXCR7 is necessary to effectively hinder CXCL12-γ-induced migration. These findings support the growing importance of understanding differences in protein isoforms, and in particular their implications for cancer treatment.

Surface-templated hydrogel patterns prompt matrix-dependent migration of breast cancer cells towards chemokine-secreting cells

This paper describes a novel technique for fabricating spatially defined cell-laden collagen hydrogels, using patterned, non-adhesive polyacrylamide-coated polydimethylsiloxane (PDMS) surfaces as a template. Precisely patterned embedded co-cultures of breast cancer cells and chemokine-producing cells generated with this technique revealed matrix-dependent and chemokine isoform-dependent migration of cancer cells. CXCL12 chemokine-secreting cells induce significantly more chemotaxis of cancer cells when the 3-D extracellular matrix (ECM) includes components that bind the secreted CXCL12 chemokines. Experimental observations using cells that secrete CXCL12 isoforms with different matrix affinities together with computational simulations show that stronger ligand–matrix interactions sharpen chemoattractant gradients, leading to increased chemotaxis of the CXCL12 gradient-sensing CXCR4 receptor-expressing (CXCR4+) cells patterned in the hydrogel. These results extend our recent report on CXCL12 isoform-dependent chemotaxis studies from 2-D to 3-D environments and additionally reveal the important role of ECM composition. The developed technology is simple, versatile and robust; and as chemoattractant-matrix interactions are common, the methods described here should be broadly applicable for study of physiological migration of many different cell types in response to a variety of chemoattractants.

A Comprehensive Analysis of CXCL12 Isoforms in Breast Cancer1,2

CXCL12-CXCR4-CXCR7 signaling promotes tumor growth and metastasis in breast cancer. Alternative splicing of CXCL12 produces isoforms with distinct structural and biochemical properties, but little is known about isoform-specific differences in breast cancer subtypes and patient outcomes. We investigated global expression profiles of the six CXCL12 isoforms, CXCR4, and CXCR7 in The Cancer Genome Atlas breast cancer cohort using next-generation RNA sequencing in 948 breast cancer and benign samples and seven breast cancer cell lines. We compared expression levels with several clinical parameters, as well as metastasis, recurrence, and overall survival (OS). CXCL12-α, -β, and -γ are highly co-expressed, with low expression correlating with more aggressive subtypes, higher stage disease, and worse clinical outcomes. CXCL12-δ did not correlate with other isoforms but was prognostic for OS and showed the same trend for metastasis and recurrence-free survival. Effects of CXCL12-δ remained independently prognostic when taking into account expression of CXCL12,CXCR4, and CXCR7. These results were also reflected when comparing CXCL12-α, -β, and -γ in breast cancer cell lines. We summarized expression of all CXCL12 isoforms in an important chemokine signaling pathway in breast cancer in a large clinical cohort and common breast cancer cell lines, establishing differences among isoforms in multiple clinical, pathologic, and molecular subgroups. We identified for the first time the clinical importance of a previously unstudied isoform, CXCL12-δ.

Microfluidic source-sink model reveals effects of biophysically distinct CXCL12 isoforms in breast cancer chemotaxis

Chemokines critically regulate chemotaxis in normal and pathologic states, but there is limited understanding of how multicellular interactions generate gradients needed for cell migration. Previous studies of chemotaxis of CXCR4+ cells toward chemokine CXCL12 suggest the requirement of cells expressing scavenger receptor CXCR7 in a source-sink system. We leveraged an established microfluidic device to discover that chemotaxis of CXCR4 cells toward distinct isoforms of CXCL12 required CXCR7 scavenging only under conditions with higher than optimal levels of CXCL12. Chemotaxis toward CXCL12-β and -γ isoforms, which have greater binding to extracellular molecules and have been largely overlooked, was less dependent on CXCR7 than the more commonly studied CXCL12-α. Chemotaxis of CXCR4+ cells toward even low levels of CXCL12-γ and CXCL12-β still occurred during treatment with a FDA-approved inhibitor of CXCR4. We also detected CXCL12-γ only in breast cancers from patients with advanced disease. Physiological gradient formation within the device facilitated interrogation of key differences in chemotaxis among CXCL12 isoforms and suggests CXCL12-γ as a biomarker for metastatic cancer.

Circulation: Clinical Summaries

<i>Circulation:</i> Clinical Summaries

Homeostatic and Tissue Reparation Defaults in Mice Carrying Selective Genetic Invalidation of CXCL12/Proteoglycan Interactions

Interaction with heparan sulfate proteoglycans is supposed to provide chemokines with the capacity to immobilize on cell surface and extracellular matrix for accomplishing both tissue homing and signaling of attracted cells. However, the consequences of the exclusive invalidation of such interaction on the roles played by endogenous chemokines in vivo remain unascertained. We engineered a mouse carrying a Cxcl12 gene (Cxcl12(Gagtm)) mutation that precludes interactions with heparan sulfate structures while not affecting CXCR4-dependent cell signaling of CXCL12 isoforms (α, β, γ). Cxcl12(Gagtm/Gagtm) mice develop normally, express normal levels of total and isoform-specific Cxcl12 mRNA, and show increased counting of circulating CD34(+) hematopoietic precursor cells. After induced acute ischemia, a marked impaired capacity to support revascularization was observed in Cxcl12(Gagtm/Gagtm) animals associated with a reduced number of infiltrating cells in the ischemic tissue despite the massive expression of CXCL12 isoforms. Importantly, exogenous administration of CXCL12γ, which binds heparan sulfate with the highest affinity ever reported for a cytokine, fully restores vascular growth, whereas heparan sulfate-binding CXCL12γ mutants failed to promote revascularization in Cxcl12(Gagtm/Gagtm) animals. These findings prove the role played by heparan sulfate interactions in the functions of CXCL12 in both homeostasis and physiopathological settings and document for the first time the paradigm of chemokine immobilization in vivo.

CXCL12γ isoform is expressed on endothelial and dendritic cells in rheumatoid arthritis synovium and regulates T cell activation

CXCL12γ is an alternative splicing isoform of CXCL12 with enhanced affinity for heparan sulfate (HS) proteoglycans. This study was undertaken to investigate the distribution and potential function of CXCL12γ in rheumatoid arthritis (RA) synovium and normal lymphoid tissue, where its immobilization to HS may be relevant in pathologic or homeostatic immune cell migration and activation. Expression of CXCL12 or CXCL12γ was immunodetected in RA and normal synovium, lymphoid tissue, and cultured cells with anti-pan-CXCL12 or anti-CXCL12γ-specific monoclonal antibodies. CXCL12α and CXCL12γ messenger RNA expression was analyzed by quantitative reverse transcription-polymerase chain reaction. Binding of wild-type CXCL12 isoforms or their HS binding-defective mutants to monocyte-derived dendritic cells (DCs) was analyzed by flow cytometry. The effect of DC-bound CXCL12α and CXCL12γ on T cell activation was analyzed in DC/T cell allogeneic cultures. CXCL12γ expression was increased in RA compared to normal synovium and preferentially located in endothelia and DC-SIGN-positive cells. This distribution was also observed in lymphoid organs. Surface-bound CXCL12γ was detected in a fraction of freshly isolated DCs. Monocyte-derived DCs, but not monocytes, showed a high capacity to bind CXCL12γ in an HS-dependent manner. Surface-bound CXCL12α and CXCL12γ on monocyte-derived DCs were potent inhibitors of allogeneic T cell activation, in contrast to the T cell-stimulatory effects of soluble CXCL12 proteins. CXCL12γ shows a specific and similar distribution in RA synovium and lymphoid tissue, consistent with its higher HS binding affinity. Presentation of CXCL12 to T cells on membrane HS in DCs can play a distinct regulatory role in T cell activation.

Developmental Expression Profile of the CXCL12γ Isoform: Insights Into its Tissue‐Specific Role

The CXCL12gamma chemokine arises by alternative splicing from Cxcl12, an highly conserved gene that plays pivotal, non-redundant roles during development. The interaction of the highly cationic carboxy-terminal (C-ter) domain of CXCL12gamma with glycosaminoglycans (GAG) critically determines the biological properties of this chemokine. Indeed, CXCL12gamma isoform displays sustained in vivo recruitment of leukocytes and endothelial progenitor cells as compared to other CXCL12 isoforms. Despite the important, specific roles of CXCL12gamma in vivo, the current knowledge about its distribution in embryo and adult tissues is scarce. In this study, we have characterized by both RT-PCR and immunohistochemistry the expression profile and tissue distribution of CXCL12gamma, which showed a distinct mRNA expression pattern during organogenesis that correlates with the specific expression of the CXCL12 gamma protein in several tissues and cell types during development. Our results support the biological relevance of CXCL12 gamma in vivo, and shed light on the specific roles that this novel isoform could play in muscle development and vascularization as well as on the regulation of essential homeostatic functions during the embryonic development.

Citrullination of CXCL12 Differentially Reduces CXCR4 and CXCR7 Binding with Loss of Inflammatory and Anti-HIV-1 Activity via CXCR4

Posttranslational proteolytic processing of chemokines is a natural mechanism to regulate inflammation. In this study, we describe modification of the CXC chemokine stromal cell-derived factor 1alpha/CXCL12 by peptidylarginine deiminase (PAD) that converts arginine residues into citrulline (Cit), thereby reducing the number of positive charges. The three NH(2)-terminal arginines of CXCL12, Arg(8), Arg(12), and Arg(20), were citrullinated upon incubation with PAD. The physiologic relevance of citrullination was demonstrated by showing coexpression of CXCL12 and PAD in Crohn's disease. Three CXCL12 isoforms were synthesized for biologic characterization: CXCL12-1Cit, CXCL12-3Cit, and CXCL12-5Cit, in which Arg(8), Arg(8)/Arg(12)/Arg(20), or all five arginines were citrullinated, respectively. Replacement of only Arg(8) caused already impaired (30-fold reduction) CXCR4 binding and signaling (calcium mobilization, phosphorylation of ERK and protein kinase B) properties. Interaction with CXCR4 was completely abolished for CXCL12-3Cit and CXCL12-5Cit. However, the CXCR7-binding capacities of CXCL12-1Cit and CXCL12-3Cit were, respectively, intact and reduced, whereas CXCL12-5Cit failed to bind CXCR7. In chemotaxis assays with lymphocytes and monocytes, CXCL12-3Cit and CXCL12-5Cit were completely devoid of activity, whereas CXCL12-1Cit, albeit at higher concentrations than CXCL12, induced migration. The antiviral potency of CXCL12-1Cit was reduced compared with CXCL12 and CXCL12-3Cit and CXCL12-5Cit (maximal dose 200 nM) could not inhibit infection of lymphocytic MT-4 cells with the HIV-1 strains NL4.3 and HE. In conclusion, modification of CXCL12 by one Cit severely impaired the CXCR4-mediated biologic effects of this chemokine and maximally citrullinated CXCL12 was inactive. Therefore, PAD is a potent physiologic down-regulator of CXCL12 function.

The CXCL12γ Chemokine Displays Unprecedented Structural and Functional Properties that Make It a Paradigm of Chemoattractant Proteins

The CXCL12γ chemokine arises by alternative splicing from Cxcl12, an essential gene during development. This protein binds CXCR4 and displays an exceptional degree of conservation (99%) in mammals. CXCL12γ is formed by a protein core shared by all CXCL12 isoforms, extended by a highly cationic carboxy-terminal (C-ter) domain that encompass four overlapped BBXB heparan sulfate (HS)-binding motifs. We hypothesize that this unusual domain could critically determine the biological properties of CXCL12γ through its interaction to, and regulation by extracellular glycosaminoglycans (GAG) and HS in particular. By both RT-PCR and immunohistochemistry, we mapped the localization of CXCL12γ both in mouse and human tissues, where it showed discrete differential expression. As an unprecedented feature among chemokines, the secreted CXCL12γ strongly interacted with cell membrane GAG, thus remaining mostly adsorbed on the plasmatic membrane upon secretion. Affinity chromatography and surface plasmon resonance allowed us to determine for CXCL12γ one of the higher affinity for HS (Kd = 0.9 nM) ever reported for a protein. This property relies in the presence of four canonical HS-binding sites located at the C-ter domain but requires the collaboration of a HS-binding site located in the core of the protein. Interestingly, and despite reduced agonist potency on CXCR4, the sustained binding of CXCL12γ to HS enabled it to promote in vivo intraperitoneal leukocyte accumulation and angiogenesis in matrigel plugs with much higher efficiency than CXCL12α. In good agreement, mutant CXCL12γ chemokines selectively devoid of HS-binding capacity failed to promote in vivo significant cell recruitment. We conclude that CXCL12γ features unique structural and functional properties among chemokines which rely on the presence of a distinctive C-ter domain. The unsurpassed capacity to bind to HS on the extracellular matrix would make CXCL12γ the paradigm of haptotactic proteins, which regulate essential homeostatic functions by promoting directional migration and selective tissue homing of cells.