To our best knowledge this work is the first in vivo description of protein expression of multiple ZIPs and MTs in human microvasculature. A complete list analysis of the ZIPs and MTs, and the third family of zinc regulation proteins, SLC30As/ZnTs remains however a task for future investigations. Despite a limitation that expression at the gene level was not analysed, protein expression of multiple members of ZIPs and MTs supports a hypothesis that there is a redundancy in zinc regulation system which may be required for a tight control of zinc homeostasis in vascular functions. As another limitation, immunofluorescence results should be interpreted with caution on antibodies’ specificity; to minimize this, most of the primary antibodies used in this study were tested independently by Western blots or immunogen/antibody competition in our previous publications, or published by the manufacturers.
Results of this study are mostly in line with our previous data on gene expression in primary cell culture (Abdo et al. 2021), with exceptions as discussed below. Thus, while abundant mRNA expression was found in primary cell cultures for ZIP6 and ZIP9, their protein expression in this study could not be detected in microvessels from subcutaneous biopsies. Furthermore, while the relative abundance of gene expression in cell cultures was low for ZIP2, ZIP12 and MT3, protein immunoreactivities in microvascular tissues were varying, brighter in subpopulation(s) of microvessels. Apart from a potential sensitivity issue that could not be ruled out, one possible cause was that while mRNA data was obtained from primary cells of aorta and pulmonary artery, protein expression data was from in vivo sampling of microvessels. Furthermore, while primary cell culture data reflected a normal state in which vascular cells expressed minimal levels of ZIP2, ZIP12 and MT3, biopsies included pathological conditions that could induce these proteins. In accordance with this, previous data provides multiple evidence that ZIP12 could be induced in vivo in vasculature in human patients and rat models of PAH (Zhao et al. 2015; Tran et al. 2021; Xiao et al. 2021). In vitro, both ZIP2 and ZIP12 were shown induced at mRNA and protein levels in vascular cells by depletion of zinc (Abdo et al. 2021). The tissue localization data in this study was in line with previous studies (Zhao et al. 2015; Abdo et al. 2021; Tran et al. 2021); ZIP12 was localized to both the endothelial and smooth muscle cell types of the vascular wall. Microvascular expression of ZIP14 was consistent with previous data, that influx of labile zinc in cultured sheep pulmonary artery endothelial cells was sensitive to ZIP14 siRNA indicating the presence of functional ZIP14 in this cell type (Thambiayya et al. 2012). Our findings of highly expressed ZIP1 in microvessels was in line with a notion that ZIP1 is expressed ubiquitously across cell types (Schweigel-Röntgen 2014). Regarding other ZIPs, to our knowledge ours is the first study to examine their localization in vascular walls.
Known as free radical scavengers, MTs are surrogate markers of oxidative stress and indicators of labile intracellular zinc levels (Mareiro et al. 2017). MTs have been studied in various cell culture models of vascular endothelial cells in oxidative stress associated with exposure to heavy metals, or other stress stimuli (Kaji et al. 1993; Conway et al. 2010; Thambiayya et al. 2012; Fujie et al. 2020; Rubiolo et al. 2021). Metallothioneins have also been commonly reported to be elevated in PAH patients as well as in experimental models of PAH (Maarman 2018). In vitro studies showed that MT can respond to nitride oxide (free radical and vasodilator mediator) by releasing Zn2 + ions (Kroncke et al. 1994; Thambiayya et al. 2012), which could be relevant to a mechanism of vasodilation. Surprisingly MT expression and functions in the other vascular cell type namely smooth muscle have been paid little attention. In a rare report, using immunohistochemical staining and immunogold electron microscopy, it was noted that most of the MT induced in human atherosclerotic lesions was localized to the vascular smooth muscle cells (Göbel et al. 2000). Our finding of smooth muscle as the major harbour of MTs in the microvascular wall further puts this cell type in the spotlight of future investigations into vascular zinc biology.
As a rationale for differential analysis of quantitative data in muscularized vs. non-muscularized microvessels, increased proliferation (i.e. exit from the normally quiescent state) of smooth muscle is considered a key process in vascular pathology, e.g. in atherosclerosis (Sedding et al. 2018) and diabetic vascular restenosis (Moshapa et al. 2019). In studies of PAH, increased levels of lung microvascular muscularization serve as an indicator of pathologic changes leading to resistance and increased pulmonary blood pressure (Zhao et al. 2015; Harper et al. 2019; Maietta et al. 2021). In this study, the large heterogeneity of expression of zinc regulation proteins in subcutaneous microvessels was found to be associated with their state of muscularization, in particular for ZIP1, ZIP2, ZIP12 and MT3. The difference in muscularization could at least partially be contributed by arteriole-vs-venule difference, which could not be accessed without exact localization of microvessels prior or posterior to capillary circulation. Therefore, we could not precisely ascribe the upregulated expression of the above-mentioned proteins to arterioles vs. venules.
The differences between the two microvessels subpopulations in their expression of vascular-active molecules, however, give a notable indication on functional differentiation, in addition to morphologic features. Known primarily as a potent vasoconstrictor, ET-1 has broad effects on various pathways critical for vascular functions and diseases e.g. induction of VCAM-1 (Ishizuka 1999), pro-inflammatory activation of leucocytes including neutrophils (Kaszaki et al., 2008) and macrophages (Zhang et al. 2021). Relevant to the vascular pathology, ET-1 was reported to activate smooth muscle by stimulating protein synthesis, promoting proliferation and hypertrophy of pulmonary arterial smooth muscle cells (Chua et al. 1992; Zamora et al. 1993). In this study increased particulate immunofluorescence of ET-1 in muscularized microvessels was localized to endothelial-smooth muscle junctions, supporting a hypothesis that paracrine ET-1-mediated endothelial-smooth muscle crosstalk may be required for not only vasoconstriction, but also proliferation of vascular smooth muscle. As a marker of both endothelial-mesenchymal transition and vascular remodelling, increased α-SMA expression is known to be associated with exposure to mechanical stress that could also induce activation of p38 MAPK (Wang et al. 2006). In a study of mechanisms leading to PAH, activation of p38 MAPK was found to be associated with oxidative stress and inflammation (Church et al. 2015). Thus, data presented here argues that upregulation of zinc regulation proteins ZIP1, ZIP2, ZIP2 and MT3 is associated with a functionally activated state, compared to a relatively quiescent state of microvessels.