Endogenous l- to d-amino acid residue isomerization modulates selectivity between distinct neuropeptide receptor family members.

The l- to d-amino acid residue isomerization of neuropeptides is an understudied post-translational modification found in animals across several phyla. Despite its physiological importance, little information is available regarding the impact of endogenous peptide isomerization on receptor recognition and activation. As a result, the full roles peptide isomerization play in biology are not well understood. Here, we identify that the Aplysia allatotropin-related peptide (ATRP) signaling system utilizes l- to d-residue isomerization of one amino acid residue in the neuropeptide ligand to modulate selectivity between two distinct G protein-coupled receptors (GPCRs). We first identified a novel receptor for ATRP that is selective for the D2-ATRP form, which bears a single d-phenylalanine residue at position 2. Using cell-based receptor activation experiments, we then characterized the stereoselectivity of the two known ATRP receptors for both endogenous ATRP diastereomers, as well as for homologous toxin peptides from a carnivorous predator. We found that the ATRP system displayed dual signaling through both the Gαq and Gαs pathways, and each receptor was selectively activated by one naturally occurring ligand diastereomer over the other. Overall, our results provide insights into an unexplored mechanism by which nature regulates intercellular communication. Given the challenges in detecting l- to d-residue isomerization from complex mixtures de novo and in identifying receptors for novel neuropeptides, it is likely that other neuropeptide-receptor systems may also utilize changes in stereochemistry to modulate receptor selectivity in a manner similar to that discovered here.

Two C-terminal isoforms of Aplysia tachykinin-related peptide receptors exhibit phosphorylation-dependent and phosphorylation-independent desensitization mechanisms.

Diversity, a hallmark of G protein-coupled receptor (GPCR) signaling, partly stems from alternative splicing of a single gene generating more than one isoform for a receptor. Additionally, receptor responses to ligands can be attenuated by desensitization upon prolonged or repeated ligand exposure. Both phenomena have been demonstrated and exemplified by the deuterostome tachykinin signaling system, although the role of phosphorylation in desensitization remains a subject of debate. Here, we describe the signaling system for tachykinin-related peptides (TKRPs) in a protostome, mollusk Aplysia. We cloned the Aplysia TKRP precursor, which encodes three TKRPs (apTKRP-1, apTKRP-2a, and apTKRP-2b) containing the FXGXR-amide motif. In situ hybridization and immunohistochemistry showed predominant expression of TKRP mRNA and peptide in the cerebral ganglia. TKRPs and their posttranslational modifications were observed in extracts of central nervous system ganglia using mass spectrometry. We identified two Aplysia TKRP receptors (apTKRPRs), named apTKRPR-A and apTKRPR-B. These receptors are two isoforms generated through alternative splicing of the same gene and differ only in their intracellular C termini. Structure-activity relationship analysis of apTKRP-2b revealed that both C-terminal amidation and conserved residues of the ligand are critical for receptor activation. C-terminal truncates and mutants of apTKRPRs suggested that there is a C-terminal phosphorylation-independent desensitization for both receptors. Moreover, apTKRPR-B also exhibits phosphorylation-dependent desensitization through the phosphorylation of C-terminal Ser/Thr residues. This comprehensive characterization of the Aplysia TKRP signaling system underscores the evolutionary conservation of the TKRP and TK signaling systems, while highlighting the intricacies of receptor regulation through alternative splicing and differential desensitization mechanisms.

Mass Spectrometry Approaches Empowering Neuropeptide Discovery and Therapeutics.

The discovery of insulin in the early 1900s ushered in the era of research related to peptides acting as hormones and neuromodulators, among other regulatory roles. These essential gene products are found in all organisms, from the most primitive to the most evolved, and carry important biologic information that coordinates complex physiology and behavior; their misregulation has been implicated in a variety of diseases. The evolutionary origins of at least 30 neuropeptide signaling systems have been traced to the common ancestor of protostomes and deuterostomes. With the use of relevant animal models and modern technologies, we can gain mechanistic insight into orthologous and paralogous endogenous peptides and translate that knowledge into medically relevant insights and new treatments. Groundbreaking advances in medicine and basic science influence how signaling peptides are defined today. The precise mechanistic pathways for over 100 endogenous peptides in mammals are now known and have laid the foundation for multiple drug development pipelines. Peptide biologics have become valuable drugs due to their unique specificity and biologic activity, lack of toxic metabolites, and minimal undesirable interactions. This review outlines modern technologies that enable neuropeptide discovery and characterization, and highlights lessons from nature made possible by neuropeptide research in relevant animal models that is being adopted by the pharmaceutical industry. We conclude with a brief overview of approaches/strategies for effective development of peptides as drugs. SIGNIFICANCE STATEMENT: Neuropeptides, an important class of cell-cell signaling molecules, are involved in maintaining a range of physiological functions. Since the discovery of insulin's activity, over 100 bioactive peptides and peptide analogs have been used as therapeutics. Because these are complex molecules not easily predicted from a genome and their activity can change with subtle chemical modifications, mass spectrometry (MS) has significantly empowered peptide discovery and characterization. This review highlights contributions of MS-based research towards the development of therapeutic peptides.

Proteomic Identification of Seasonally Expressed Proteins Contributing to Heart Function and the Avoidance of Skeletal Muscle Disuse Atrophy in a Hibernating Mammal.

Hibernation in the thirteen-lined ground squirrel (Ictidomys tridecemlineatus) takes place over 4-6 months and is characterized by multiday bouts of hypothermic torpor (5-7 °C core body temperature) that are regularly interrupted every 1-2 weeks by brief (12-24 h) normothermic active periods called interbout arousals. Our goal was to gain insight into the molecular mechanisms that underlie the hibernator's ability to preserve heart function and avoid the deleterious effects of skeletal muscle disuse atrophy over prolonged periods of inactivity, starvation, and near-freezing body temperatures. To achieve this goal, we performed organelle enrichment of heart and skeletal muscle at five seasonal time points followed by LC-MS-based label-free quantitative proteomics. In both organs, we saw an increase in the levels of many proteins as ground squirrels transition from an active state to a prehibernation state in the fall. Interestingly, seasonal abundance patterns identified DHRS7C, SRL, TRIM72, RTN2, and MPZ as potential protein candidates for mitigating disuse atrophy in skeletal muscle, and ex vivo contractile mechanics analysis revealed no deleterious effects in the ground squirrel's muscles despite prolonged sedentary activity. Overall, an increased understanding of protein abundance in hibernators may enable novel therapeutic strategies to treat muscle disuse atrophy and heart disease in humans.

Affinity-Driven Aryl Diazonium Labeling of Peptide Receptors on Living Cells.

Peptide-receptor interactions play critical roles in a wide variety of physiological processes. Methods to link bioactive peptides covalently to unmodified receptors on the surfaces of living cells are valuable for studying receptor signaling, dynamics, and trafficking and for identifying novel peptide-receptor interactions. Here, we utilize peptide analogues bearing deactivated aryl diazonium groups for the affinity-driven labeling of unmodified receptors. We demonstrate that aryl diazonium-bearing peptide analogues can covalently label receptors on the surface of living cells using both the neurotensin and the glucagon-like peptide 1 receptor systems. Receptor labeling occurs in the complex environment of the cell surface in a sequence-specific manner. We further demonstrate the utility of this covalent labeling approach for the visualization of peptide receptors by confocal fluorescence microscopy and for the enrichment and identification of labeled receptors by mass spectrometry-based proteomics. Aryl diazonium-based affinity-driven receptor labeling is attractive due to the high abundance of tyrosine and histidine residues susceptible to azo coupling in the peptide binding sites of receptors, the ease of incorporation of aryl diazonium groups into peptides, and the relatively small size of the aryl diazonium group. This approach should prove to be a powerful and relatively general method to study peptide-receptor interactions in cellular contexts.

AI protein structure prediction-based modeling and mutagenesis of a protostome receptor and peptide ligands reveal key residues for their interaction.

The protostome leucokinin (LK) signaling system, including LK peptides and their G protein-coupled receptors, has been characterized in several species. Despite the progress, molecular mechanisms governing LK peptide-receptor interactions remain to be elucidated. Previously, we identified a precursor protein for Aplysia leucokinin-like peptides (ALKs) that contains the greatest number of amidated peptides among LK precursors in all species identified so far. Here, we identified the first ALK receptor from Aplysia, ALKR. We used cell-based IP1 activation assays to demonstrate that two ALK peptides with the most copies, ALK1 and ALK2, activated ALKR with high potencies. Other endogenous ALK-derived peptides bearing the FXXWX-amide motif also activated ALKR to various degrees. Our examination of cross-species activity of ALKs with the Anopheles LK receptor was consistent with a critical role for the FXXWX-amide motif in receptor activity. Furthermore, we showed, through alanine substitution of ALK1, the highly conserved phenylalanine (F), tryptophan (W), and C-terminal amidation were each essential for receptor activation. Finally, we used an artificial intelligence-based protein structure prediction server (Robetta) and Autodock Vina to predict the ligand-bound conformation of ALKR. Our model predicted several interactions (i.e., hydrophobic interactions, hydrogen bonds, and amide-pi stacking) between ALK peptides and ALKR, and several of our substitution and mutagenesis experiments were consistent with the predicted model. In conclusion, our results provide important information defining possible interactions between ALK peptides and their receptors. The workflow utilized here may be useful for studying other ligand-receptor interactions for a neuropeptide signaling system, particularly in protostomes.

Differential membrane binding of α/ß-peptide foldamers: implications for cellular delivery and mitochondrial targeting.

The intrinsic pathway of apoptosis is regulated by the Bcl-2 family of proteins. Inhibition of the anti-apoptotic members represents a strategy to induce apoptotic cell death in cancer cells. We have measured the membrane binding properties of a series of peptides, including modified α/ß-peptides, designed to exhibit enhanced membrane permeability to allow cell entry and improved access for engagement of Bcl-2 family members. The peptide cargo is based on the pro-apoptotic protein Bim, which interacts with all anti-apoptotic proteins to initiate apoptosis. The α/ß-peptides contained cyclic ß-amino acid residues designed to increase their stability and membrane-permeability. Dual polarisation interferometry was used to study the binding of each peptide to two different model membrane systems designed to mimic either the plasma membrane or the outer mitochondrial membrane. The impact of each peptide on the model membrane structure was also investigated, and the results demonstrated that the modified peptides had increased affinity for the mitochondrial membrane and significantly altered the structure of the bilayer. The results also showed that the presence of an RRR motif significantly enhanced the ability of the peptides to bind to and insert into the mitochondrial membrane mimic, and provide insights into the role of selective membrane targeting of peptides.

Trimer-to-Monomer Disruption Mechanism for a Potent, Protease-Resistant Antagonist of Tumor Necrosis Factor-α Signaling.

Aberrant tumor necrosis factor-α (TNFα) signaling is associated with many inflammatory diseases. The homotrimeric quaternary structure of TNFα is essential for receptor recognition and signal transduction. Previously, we described an engineered α/ß-peptide inhibitor that potently suppresses TNFα activity and resists proteolysis. Here, we present structural evidence that both the α/ß-peptide inhibitor and an all-α analogue bind to a monomeric form of TNFα. Calorimetry data support a 1:1 inhibitor/TNFα stoichiometry in solution. In contrast, previous cocrystal structures involving peptide or small-molecule inhibitors have shown the antagonists engaging a TNFα dimer. The structural data reveal why our inhibitors favor monomeric TNFα. Previous efforts to block TNFα-induced cell death with peptide inhibitors revealed that surfactant additives to the assay conditions cause a more rapid manifestation of inhibitory activity than is observed in the absence of additives. We attributed this effect to a loose surfactant TNFα association that lowers the barrier to trimer dissociation. Here, we used the new structural data to design peptide inhibitors bearing a surfactant-inspired appendage intended to facilitate TNFα trimer dissociation. The appendage modified the time course of protection from cell death.

Aplysia allatotropin-related peptide and its newly identified d-amino acid-containing epimer both activate a receptor and a neuronal target.

l- to d-residue isomerization is a post-translational modification (PTM) present in neuropeptides, peptide hormones, and peptide toxins from several animals. In most cases, the d-residue is critical for the biological function of the resulting d-amino acid-containing peptide (DAACP). Here, we provide an example in native neuropeptides in which the DAACP and its all-l-amino acid epimer are both active at their newly identified receptor in vitro and at a neuronal target associated with feeding behavior. On the basis of sequence similarity to a known DAACP from cone snail venom, we hypothesized that allatotropin-related peptide (ATRP), a neuropeptide from the neuroscience model organism Aplysia californica, may form multiple diastereomers in the Aplysia central nervous system. We determined that ATRP exists as a d-amino acid-containing peptide (d2-ATRP) and identified a specific G protein-coupled receptor as an ATRP receptor. Interestingly, unlike many previously reported DAACPs and their all-l-residue analogs, both l-ATRP and d2-ATRP were potent agonists of this receptor and active in electrophysiological experiments. Finally, d2-ATRP was much more stable than its all-l-residue counterpart in Aplysia plasma, suggesting that in the case of ATRP, the primary role of the l- to d-residue isomerization may be to protect this peptide from aminopeptidase activity in the extracellular space. Our results indicate that l- to d-residue isomerization can occur even in an all-l-residue peptide with a known biological activity and that in some cases, this PTM may help modulate peptide signal lifetime in the extracellular space rather than activity at the cognate receptor.

Conformational investigation of the structure-activity relationship of GdFFD and its analogues on an achatin-like neuropeptide receptor of Aplysia californica involved in the feeding circuit.

Proteins and peptides in nature are almost exclusively made from l-amino acids, and this is even more absolute in the metazoan. With the advent of modern bioanalytical techniques, however, previously unappreciated roles for d-amino acids in biological processes have been revealed. Over 30 d-amino acid containing peptides (DAACPs) have been discovered in animals where at least one l-residue has been isomerized to the d-form via an enzyme-catalyzed process. In Aplysia californica, GdFFD and GdYFD (the lower-case letter "d" indicates a d-amino acid residue) modulate the feeding behavior by activating the Aplysia achatin-like neuropeptide receptor (apALNR). However, little is known about how the three-dimensional conformation of DAACPs influences activity at the receptor, and the role that d-residues play in these peptide conformations. Here, we use a combination of computational modeling, drift-tube ion-mobility mass spectrometry, and receptor activation assays to create a simple model that predicts bioactivities for a series of GdFFD analogs. Our results suggest that the active conformations of GdFFD and GdYFD are similar to their lowest energy conformations in solution. Our model helps connect the predicted structures of GdFFD analogs to their activities, and highlights a steric effect on peptide activity at position 1 on the GdFFD receptor apALNR. Overall, these methods allow us to understand ligand-receptor interactions in the absence of high-resolution structural data.

Targeting diverse protein-protein interaction interfaces with α/ß-peptides derived from the Z-domain scaffold.

Peptide-based agents derived from well-defined scaffolds offer an alternative to antibodies for selective and high-affinity recognition of large and topologically complex protein surfaces. Here, we describe a strategy for designing oligomers containing both α- and ß-amino acid residues ("α/ß-peptides") that mimic several peptides derived from the three-helix bundle "Z-domain" scaffold. We show that α/ß-peptides derived from a Z-domain peptide targeting vascular endothelial growth factor (VEGF) can structurally and functionally mimic the binding surface of the parent peptide while exhibiting significantly decreased susceptibility to proteolysis. The tightest VEGF-binding α/ß-peptide inhibits the VEGF165-induced proliferation of human umbilical vein endothelial cells. We demonstrate the versatility of this strategy by showing how principles underlying VEGF signaling inhibitors can be rapidly extended to produce Z-domain-mimetic α/ß-peptides that bind to two other protein partners, IgG and tumor necrosis factor-α. Because well-established selection techniques can identify high-affinity Z-domain derivatives from large DNA-encoded libraries, our findings should enable the design of biostable α/ß-peptides that bind tightly and specifically to diverse targets of biomedical interest. Such reagents would be useful for diagnostic and therapeutic applications.

Iterative Nonproteinogenic Residue Incorporation Yields α/ß-Peptides with a Helix-Loop-Helix Tertiary Structure and High Affinity for VEGF.

Inhibition of specific protein-protein interactions is attractive for a range of therapeutic applications, but the large and irregularly shaped contact surfaces involved in many such interactions make it challenging to design synthetic antagonists. Here, we describe the development of backbone-modified peptides containing both α- and ß-amino acid residues (α/ß-peptides) that target the receptor-binding surface of vascular endothelial growth factor (VEGF). Our approach is based on the Z-domain, which adopts a three-helix bundle tertiary structure. We show how a two-helix "mini-Z-domain" can be modified to contain ß and other nonproteinogenic residues while retaining the target-binding epitope by using iterative unnatural residue incorporation. The resulting α/ß-peptides are less susceptible to proteolysis than is their parent α-peptide, and some of these α/ß-peptides match the full-length Z-domain in terms of affinity for receptor-recognition surfaces on the VEGF homodimer.

α/ß-Peptide Foldamers Targeting Intracellular Protein-Protein Interactions with Activity in Living Cells.

Peptides can be developed as effective antagonists of protein-protein interactions, but conventional peptides (i.e., oligomers of l-α-amino acids) suffer from significant limitations in vivo. Short half-lives due to rapid proteolytic degradation and an inability to cross cell membranes often preclude biological applications of peptides. Oligomers that contain both α- and ß-amino acid residues ("α/ß-peptides") manifest decreased susceptibility to proteolytic degradation, and when properly designed these unnatural oligomers can mimic the protein-recognition properties of analogous "α-peptides". This report documents an extension of the α/ß-peptide approach to target intracellular protein-protein interactions. Specifically, we have generated α/ß-peptides based on a "stapled" Bim BH3 α-peptide, which contains a hydrocarbon cross-link to enhance α-helix stability. We show that a stapled α/ß-peptide can structurally and functionally mimic the parent stapled α-peptide in its ability to enter certain types of cells and block protein-protein interactions associated with apoptotic signaling. However, the α/ß-peptide is nearly 100-fold more resistant to proteolysis than is the parent stapled α-peptide. These results show that backbone modification, a strategy that has received relatively little attention in terms of peptide engineering for biomedical applications, can be combined with more commonly deployed peripheral modifications such as side chain cross-linking to produce synergistic benefits.

Label-Free Quantitation of Endogenous Peptides.

Liquid chromatography-mass spectrometry (LC-MS)-based peptidomics methods allow for the detection and identification of many peptides in a complex biological mixture in an untargeted manner. Quantitative peptidomics approaches allow for comparisons of peptide abundance between different samples, allowing one to draw conclusions about peptide differences as a function of experimental treatment or physiology. While stable isotope labeling is a powerful approach for quantitative proteomics and peptidomics, advances in mass spectrometry instrumentation and analysis tools have allowed label-free methods to gain popularity in recent years. In a general label-free quantitative peptidomics experiment, peak intensity information for each peptide is compared across multiple LC-MS runs. Here, we outline a general approach for label-free quantitative peptidomics experiments, including steps for sample preparation, LC-MS data acquisition, data processing, and statistical analysis. Special attention is paid to address run-to-run variability, which can lead to several major problems in label-free experiments. Overall, our method provides researchers with a framework for the development of their own quantitative peptidomics workflows applicable to quantitation of peptides from a wide variety of different biological sources.

Nontargeted Identification of D-Amino Acid-Containing Peptides Through Enzymatic Screening, Chiral Amino Acid Analysis, and LC-MS.

D-amino acid-containing peptides (DAACPs) in animals are a class of bioactive molecules formed via the posttranslational modification of peptides consisting of all-L-amino acid residues. Amino acid residue isomerization greatly impacts the function of the resulting DAACP. However, because isomerization does not change the peptide's mass, this modification is difficult to detect by most mass spectrometry-based peptidomic approaches. Here we describe a method for the identification of DAACPs that can be used to systematically survey peptides extracted from a tissue sample in a nontargeted manner.

Structure-guided rational design of α/ß-peptide foldamers with high affinity for BCL-2 family prosurvival proteins.

We have used computational methods to improve the affinity of a foldamer ligand for its target protein. The effort began with a previously reported α/ß-peptide based on the BH3 domain of the proapoptotic protein Puma; this foldamer binds tightly to Bcl-x(L) but weakly to Mcl-1. The crystal structure of the Puma-derived α/ß-peptide complexed to Bcl-x(L) was used as the basis for computational design of variants intended to display improved binding to Mcl-1. Molecular modelling suggested modification of three α residues of the original α/ß backbone. Individually, each substitution caused only a modest (4- to 15-fold) gain in affinity; however, together the three substitutions led to a 250-fold increase in binding to Mcl-1. These modifications had very little effect on affinity for Bcl-x(L). Crystal structures of a number of the new α/ß-peptides bound to either Mcl-1 or Bcl-x(L) validated the selection of each substitution. Overall, our findings demonstrate that structure-guided rational design can be used to improve affinity and alter partner selectivity of peptidic ligands with unnatural backbones that bind to specific protein partners.

Identifying and Measuring Endogenous Peptides through Peptidomics.

Endogenous peptides, such as neuropeptides and peptide hormones, play important roles in intercellular communication, can provide information on physiology, and are potential sources of biomarkers. Mass spectrometry-based peptidomics methods are underutilized tools to identify and measure endogenous peptides in a relatively nontargeted manner. The purpose of this Viewpoint is to serve as a brief introduction to the field of peptidomics so that researchers interested in studying endogenous peptides are aware of this powerful approach and can consider its application.

Peptidomic Analysis Reveals Seasonal Neuropeptide and Peptide Hormone Changes in the Hypothalamus and Pituitary of a Hibernating Mammal.

During the winter, hibernating mammals undergo extreme changes in physiology, which allow them to survive several months without access to food. These animals enter a state of torpor, which is characterized by decreased metabolism, near-freezing body temperatures, and a dramatically reduced heart rate. The neurochemical basis of this regulation is largely unknown. Based on prior evidence suggesting that the peptide-rich hypothalamus plays critical roles in hibernation, we hypothesized that changes in specific cell-cell signaling peptides (neuropeptides and peptide hormones) underlie physiological changes during torpor/arousal cycles. To test this hypothesis, we used a mass spectrometry-based peptidomics approach to examine seasonal changes of endogenous peptides that occur in the hypothalamus and pituitary of a model hibernating mammal, the thirteen-lined ground squirrel (Ictidomys tridecemlineatus). In the pituitary, we observed changes in several distinct peptide hormones as animals prepared for torpor in October, exited torpor in March, and progressed from spring (March) to fall (August). In the hypothalamus, we observed an overall increase in neuropeptides in October (pre-torpor), a decrease as the animal entered torpor, and an increase in a subset of neuropeptides during normothermic interbout arousals. Notable changes were observed for feeding regulatory peptides, opioid peptides, and several peptides without well-established functions. Overall, our study provides critical insight into changes in endogenous peptides in the hypothalamus and pituitary during mammalian hibernation that were not available from transcriptomic measurements. Understanding the molecular basis of the hibernation phenotype may pave the way for future efforts to employ hibernation-like strategies for organ preservation, combating obesity, and treatment of stroke.

Effects of Anesthetic Administration on Rat Hypothalamus and Cerebral Cortex Peptidome.

Prohormone-derived neuropeptides act as cell-cell signaling molecules to mediate a wide variety of biological processes in the animal brain. Mass spectrometry-based peptidomic experiments are valuable approaches to gain insight into the dynamics of individual peptides under different physiological conditions or experimental treatments. However, the use of anesthetics during animal procedures may confound experimental peptide measurements, especially in the brain, where anesthetics act. Here, we investigated the effects of the commonly used anesthetics isoflurane and sodium pentobarbital on the peptide profile in the rodent hypothalamus and cerebral cortex, as assessed by label-free quantitative peptidomics. Our results showed that neither anesthetic dramatically alters peptide levels, although extended isoflurane exposure did cause changes in a small number of prohormone-derived peptides in the cerebral cortex. Overall, our results demonstrate that acute anesthetic administration can be utilized in peptidomic experiments of the hypothalamus and cerebral cortex without greatly affecting the measured peptide profiles.

Extending foldamer design beyond α-helix mimicry: α/ß-peptide inhibitors of vascular endothelial growth factor signaling.

Diverse strategies have been explored to mimic the surface displayed by an α-helical segment of a protein, with the goal of creating inhibitors of helix-mediated protein-protein interactions. Many recognition surfaces on proteins, however, are topologically more complex and less regular than a single α-helix. We describe efforts to develop peptidic foldamers that bind to the irregular receptor-recognition surface of vascular endothelial growth factor (VEGF). Our approach begins with a 19-residue α-peptide previously reported by Fairbrother et al. (Biochemistry 1998, 37, 17754) to bind to this surface on VEGF. Systematic evaluation of αâ†’ß replacements throughout this 19-mer sequence enabled us to identify homologues that contain up to ~30% ß residues, retain significant affinity for VEGF, and display substantial resistance to proteolysis. These α/ß-peptides can block VEGF-stimulated proliferation of human umbilical vein endothelial cells.