TGF-β in blood: a complex problem
Introduction
The transforming growth factor-β (TGF-β) superfamily is a collection of structurally related multi-functional cytokines that have been implicated in a wide range of physiological and pathological processes, including wound healing, development, oncogenesis, immunomodulation and atherosclerosis [1]. The prototypical member of this superfamily, TGF-β1, was originally identified as a growth factor for transformed cells, but was first purified to homogeneity from human platelets [2]. There are now known to be three closely related mammalian TGF-β isoforms (TGF-β1, -β2 and -β3) which are thought to have similar functions, at least in vitro, although less is known about TGF-β2 and TGF-β3. Each of the three isoforms is produced as a pre-pro-protein which rapidly dimerises. After loss of the signal sequence the dimer is further processed by addition of sugar moieties to the propeptide region (known as the latency-associated peptide; LAP). In addition, there is proteolytic cleavage between the LAPs and the mature dimer (which, when released from the LAP dimer, is able to bind to the TGF-β signalling receptors). After cleavage, however, the LAP dimer usually remains non-covalently associated with the mature dimer forming a complex known as the small latent complex [3]. Either prior to secretion or in the extracellular milieu the small latent complex can bind to a wide range of other proteins forming a large number of higher molecular weight latent complexes. The best characterised of these proteins are the latent TGF-β binding protein family (LTBP1-4 and fibrillin-1 and 2) [4], [5]. For example, in platelets, the majority of the TGF-β present is thought to exist in a latent complex consisting of the LTBP-1 covalently coupled to the small latent complex. This complex is called the large latent complex.
Once in the extracellular environment (such as the extracellular matrix of solid tissues or in the blood plasma), the latent complexes, possibly associated with various accessory proteins, must be activated in order to exert their biological effects. It is assumed that this activation process involves release of the mature dimer from its association with the LAP dimer. A number of pathways are likely to participate in TGF-β activation, such as cleavage of the LAP dimer by proteases, including plasmin [6], [7], and conformational changes in the latent complex induced by binding to matrix components such as thrombospondin [8], [9] or certain integrins [10]. Once activated, the TGF-β is able to signal through cell surface receptors, of which the best studied are the type I and type II TGF-β receptors (TβRI and TβRII) which can form heteromultimers in response to ligand binding resulting in an active signalling complex which is thought to be responsible for many of the cellular responses to TGF-β [11].
TGF-β1 has a very wide range of activities in vitro. For example, TGF-β regulates important cellular functions such as rate of proliferation and production of extracellular matrix proteins by a wide range of cell types [1]. Thus, misregulation of TGF-β has been proposed to play a key role in the development of a number of diseases in which the normal adult tissue architecture is progressively lost, including scarring during wound repair [12], carcinogenesis [13], atherosclerosis [14], osteoporosis [15], [16] and neurodegenerative diseases [17].
As a result of the wide range of activities attributed to TGF-β, a number of groups have investigated whether circulating levels of TGF-β1 might be altered in various disease states. With only one exception, all of these studies agree that TGF-β1 is found at detectable levels in plasma from healthy human subjects (see Table 1). Moreover, plasma TGF-β1 concentrations markedly differed (by as much as 10-fold) in subjects suffering from various diseases, including various cancers [18], [19], [20], [21], autoimmune disorders [22], [23] and atherosclerosis [24], [25], compared with control subjects. Based on these early studies, it seems likely that plasma levels of TGF-β1 may be a useful diagnostic criteria for the presence of one or more of these dieases. In addition, it remains possible, but unproven, that altered plasma levels of this multifunctional cytokine participates in disease progression, rather than simply acting as a marker for disease status. If such a pathophysiological role for plasma TGF-β1 is proven, it could become both a prognostic indicator of future risk of disease and a target for therapeutic intervention.
Section snippets
Levels of TGF-β in normal human plasma
Despite the interest in measuring levels of TGF-β1 in plasma as a result of early reports suggesting associations between plasma TGF-β1 concentration and disease, there is still no consensus on the concentration range of TGF-β1 in normal human plasma. The levels of TGF-β1 protein in plasma from various groups of normal subjects have been measured in more than 20 studies in the literature to date, yet the mean (or median) values reported range from below 0.1 ng/ml to more than 25 ng/ml (Table 1).
Initial characterisation of the TGF-β1 complexes in human plasma
We have recently used column chromatography to begin to address the question of the molecular composition of the TGF-β1 complexes in plasma [36]. When plasma, serum or platelets are applied to an anion exchange column and then unbound material washed away, all the TGF-β1 is retained on the column (assessed using the Quantikine TGF-β1 assay). Any TGF-β1 complexes which are not detected by the Quantikine TGF-β1 ELISA are, by definition, excluded from this analysis. The bound TGF-β1 can then be
Where does plasma TGF-β come from?
There are a number of potential sources for the TGF-β1 present in normal human plasma. Although release of platelet TGF-β1 during blood sampling and plasma preparation can be excluded as an artefactual source of plasma TGF-β1, it remains possible that platelets are a source of plasma TGF-β1 in vivo, either through regulated secretion or a background level of platelet degranulation. However, comparison of the column retention times of the plasma TGF-β1 complexes with that of the platelet TGF-β1
What is the mechanism of clearance of TGF-β1 from the plasma?
When radiolabelled TGF-β1 25 kDa dimer is injected into the blood, it is rapidly lost from the circulation (half-life=∼2 min; [43], [44], [45]). Analysis of the distribution of the radioactive label demonstrated that it becomes localised to the vascular endothelium [46], possibly as a result of binding to the abundant type III TGF-β receptor, endoglin (CD105). One methodological consequence of this rapid association with the endothelium is that it is difficult to analyse any other routes of
What controls the levels of plasma TGF-β?
At present there is little quantitative data to indicate to what extent regulated synthesis or regulated clearance contribute to the control of the steady state level of plasma TGF-β1. However, we have recently obtained some indirect evidence that synthesis rate plays a part in determining steady state plasma levels of TGF-β1 protein, at least in healthy individuals. Analysis of a population of post-menopausal female twins (comparing monozygotic with dizygotic pairs) has demonstrated that
Is there any active TGF-β in normal human plasma?
A number of assays, both bioassays and ELISAs, have been described which attempt to measure the amount of active TGF-β1 in complex biological fluids, such as plasma. In some cases, assays which previously have been used to measure total TGF-β1 protein after in vitro activation of the sample (such as by transient acidification), have also been used to measure active TGF-β in the sample prior to activation. There is one key advantage to using bioassays for measuring active TGF-β1 — bioassays
Conclusions
The variability among reported levels of TGF-β1 in human plasma stems primarily from two sources — preparation of plasma samples and selection of assay methodology. Appropriate protocols are now available [26] to ensure that plasma can be prepared with minimal contamination from other blood fractions, such as platelets, and widespread adoption of these protocols should reduce the variation in values reported in different studies. However, the variability due to selection of assay methodology
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2021, Pharmacology and TherapeuticsCitation Excerpt :Combination regimens with TGFβ and PD-L1 pathway inhibitors may be therapeutically synergistic (Principe et al., 2016). Elevated plasma TGFβ can identify prognostically high-risk patients (Anscher, Kong, & Jirtle, 1998; Grainger, Mosedale, & Metcalfe, 2000; Kong et al., 1995; Tas, Karabulut, Yasasever, & Duranyildiz, 2014). In nude mice bearing subcutaneous human tumor xenografts, plasma levels of TGFβ were increased up to 37-fold compared with control mice.