Microcirculatory Inflammation: A Key Driver of Adverse Reactions to mRNA-Containing COVID-19 Vaccines

2. The mRNA–LNP-induced Inflammatory Complications and their Root Cause

Table 1 shows a list of organ-specific inflammatory symptoms or illnesses that were reported in the publicly available 3-month safety surveillance report of Pfizer [8].The advantage of using these data is that the list of AEs likely excludes symptoms caused by SARS-CoV-2 infection, as the vaccine had not been administered to infected individuals. The list was compiled using data from 52 countries, minimizing the risk of regional bias. Artificial intelligence assisted in the process by scanning over 1,200 entries for terms containing the suffix “-itis”, followed by screening to exclude redundant qualifiers such as “acute”, “chronic”, “(auto)immune”, “bacterial”, or “viral”. Thus, the list does not distinguish among acute or chronic inflammatory conditions due to bacterial or viral infections or autoimmune attack; it just shows that essentially all organs can be inflicted with a form of inflammation starting sometimes within 3 months after vaccination.

The similar kinetics of vaccine-induced inflammatory AEs listed in Table 1 (developing within days or much later, in months after vaccination) and numerous commonalties in these illnesses, such as the association with reactivation of certain viral strains (Table 2), point to a common, very fundamental immune abnormality or combination of abnormalities underlying these conditions.

A recent comprehensive theory on this conundrum attributed the phenomenon, at least in part, to plausible consequences of inherent structural and functional properties of mRNA-LNPs [1,9]. The concerns related to the design of mRNA vaccines vaccine include: (i) ribosomal synthesis of the antigen (spike protein), which fundamentally alters the natural process of antigen processing and presentation; (ii) extensive chemical modification of the mRNA, rendering the spike protein translation poorly controllable and mutation-prone upon codon–anticodon pairing; (iii) the use of a proinflammatory, fusogenic aminolipid in the LNP, which promotes widespread systemic distribution of the mRNA-LNP entailing transfection of non-target cells to produce a toxin; (iv) the chemical stabilization of the spike protein, despite its known multiorgan, polymodal toxicity; (v) the choice of LNP lipid composition with reduced nanoparticle stability in aqueous environments; (vi) the PEGylation of the LNP surface, despite PEG’s known immune reactivity and immunogenicity; and (vii) recombinant production of the mRNA, which carries a risk for DNA contamination. These features collectively predispose to collateral tissue damage caused by inflammation involving cytotoxic T cell and complement-mediated autoimmune responses. Although nucleoside modifications (e.g., pseudouridine substitution) of the mRNA reduce recognition by innate immune sensors, such as TLR3, TLR7, TLR8, and retinoic acid-inducible gene I (RIG-I) [10,11,12,13,14], residual activation of innate immunity by the modified mRNA via RIG-I-like receptors remains a possibility under certain conditions [15].

Given the multicausal and multiorgan nature of vaccine-induced inflammation, numerous pathological processes are likely involved. This review focuses on microcirculatory inflammation, especially on the endothelium, as a central driver of inflammatory AEs. The task is complicated by tissue-specific differences in microcirculatory structure and function, as described below.

3. Components and Unique Organization of Microcirculation Across Tissues

The microcirculation refers to the smallest blood vessels in the body (below ~200 micrometer), including arterioles, capillaries, and venules which collectively responsible for gas and nutrient exchange, waste removal, and immune surveillance at the tissue level. Some morphological features of cells in the microcirculation are illustrated in Figure 1.

4. Endothelial Cells in the Frontline of Systemic Inflammation

Endothelial cells, being an interface between blood and tissues, are particularly responsive to inflammatory stimuli and are often the first point of contact for systemically circulating immune mediators and nanoparticles, including mRNA-LNPs. Damage to the glycocalyx, and, hence, endothelial integrity, can precipitate secondary cascades such as coagulation, complement activation, immune cell infiltration, all of which contribute to various pathophysiological states, including inflammation, sepsis, ischemia-reperfusion injury, and vascular complications related to infections and vaccinations [25,26]. The manifestations of symptoms and illnesses vary widely across organs, reflecting not only differences in immune responses but also organ-specific anatomical factors, discussed below.

5. Distinctive Microcirculatory Architectures Across Organs and Their Impacts on Vaccine-Induced Inflammations

Although all organs share a basic microvascular blueprint, the microcirculatory networks differ markedly due to a variety of anatomical, physiological, and functional factors. These include the density of the capillary network (Figure 1A), EC ultrastructure and architecture (Figure 1B,C), and endothelial subtype based on the tightness of intercellular junctions (Figure 1D) [25,27]. Additional control is exerted by local regulatory mechanisms involving adjacent tissue cells, such as pericytes around nerve fibers (Figure 1C), smooth muscle cells in precapillary sphincters (Figure 1A), or other resistance elements that modulate blood flow in response to tissue-specific functional demands [28]. These structural and functional variations render the microcirculation a highly specialized, tissue-specific interface between the bloodstream and parenchymal cells. Consequently, they critically influence the nature and extent of inflammatory responses. In particular, the localization or systemic spread of the spike protein is strongly determined by the special structures and function of microcirculation of various organs.

To illustrate the above differences with examples, the heart’s microcirculation is characterized by an exceptionally dense capillary network providing nearly one-to-one capillary/cardiomyocyte ratio. This allows fast and efficient gas exchange and nutrient supply to the cardiac muscle fibers (Figure 2A). The high-density capillary network in the myocardium, which is tightly connected to the epicardium, the inner layer of fluid-containing pericardium [29,30] provides the structural basis for the frequent vaccine-induced coincidence of pericarditis and myocarditis, a condition referred to as myopericarditis [31,32,33].

Under such conditions the continuous capillaries with tight junctions undergo capillary remodeling, endothelial activation and fenestration (32), underlying the inflammatory symptoms.

In the brain, the ECs in continuous capillaries are connected by tight junctions, forming the blood–brain barrier (BBB), which strictly regulates molecular and cellular trafficking (Figure 2B). This anatomical configuration is consistent with the emergence of localized inflammatory processes, underlying epileptic, cognitive and other focal AEs, for example optic neuritis, Guillain–Barré syndrome, facial paresis, etc. Rinaldi, 2022 #9042}. The localized, restrained inflammation also helps explain the functional neurological disorders which are characterized by neurological symptoms without detectable abnormalities upon imaging with MRI or CT [34,35,36]. The situation is similar in the spinal cord, for example in transverse myelitis, which entails localized motor, sensory, and autonomic dysfunction.

In peripheral nerves, the endoneurium, which encases the individual nerve fibers (axons) along with their myelin sheath, contains an extensive network of capillaries also with tightly bound ECs (Figure 2C). Here, too, the inflammatory signals spread locally, along the axons and the perineurial and epineurial tissue, leading to demyelinating neuritis of different afferent or efferent nerves.

In the lung, the microcirculation comprises an extensive and highly permeable capillary network that lies in close association with the alveolar epithelium (Figure 2D). This anatomical arrangement facilitates the transmission of inflammatory signals to both type I squamous epithelial cells and type II secretory cells, leading to widespread inflammatory involvement of the respiratory tissue. Consequently, typical manifestations of vaccine-induced inflammatory adverse events in the lung include alveolar infiltrates accompanied by respiratory distress, dyspnea, chest tightness, and hypoxia [37,38].

In the kidney, the microcirculation consists of two distinct capillary networks: glomerular capillaries, which form high-pressure filtration units, and peritubular capillaries, which surround the renal tubules and facilitate selective reabsorption and urine concentration within the nephron (Figure 2E). Inflammation of the ECs in both types of capillaries explain the nephrotic syndrome characteristic of vaccine injury, manifested in acute onset edema, hypoalbuminemia, and heavy proteinuria [39,40].

In the liver, the microcirculation is organized into sinusoidal capillaries with a discontinuous endothelium, allowing free distribution of inflammatory mediators among the blood and hepatocytes. Accordingly, the symptoms of vaccine-induced hepatitis reflect common hepatocyte dysfunction, manifested in acute cholestatic liver injury (biliary obstruction), abdominal pain, pruritus, fever, fatigue, anorexia, nausea and vomiting, laboratory deviations, occasionally fatal outcome in patients with preexisting liver disease [41,42,43,44,45].

Given this diversity of AEs in different organ systems, the impact of endothelitis must be tissue-specific, likely responsive to targeted anti-inflammatory therapies.

6. The Journey of mRNA-LNPs from the Deltoid Muscle to the Sites of Inflammations

Intramuscular injection of mRNA–LNP-based vaccines is generally associated with immediate interaction with antigen-presenting cells (APCs) and other immune cells at the injection site and within the draining lymph nodes. These locations are considered the primary sites for T and B cell priming and have remained the central focus of most narratives concerning the immune mechanisms of mRNA-based vaccines [46,47,48,49]. However, it is well established that LNPs injected intramuscularly rapidly distribute throughout the body, reaching various non-immune organs and cell types, many of which can take up the nanoparticles.

Three main mechanisms have been proposed to explain how mRNA-LNPs reach the systemic circulation. One is an accidental, direct injection into a small vessel. Because the standard immunization protocol does not require aspiration or needle retraction prior to injection [50], inadvertent intravenous administration and fast entry of vaccine into the brachial vein may occur, particularly in regions with dense capillary networks, such as the deltoid muscle. Second, entry via the lymphatic system which may start through the blind-ended portions of lymphatic vessels, which are leaflet-like endothelial structures that open in response to increased interstitial pressure (Fig. 1A). The 300 µL bolus of vaccine can transiently and locally raise tissue pressure above the typically low pressure within lymphatic vessels, promoting nanoparticle entry. Once inside the lymphatic network, rhythmic contractions and relaxations of the lymphatic vessel walls propel the nanoparticles through valve-separated chambers and lymph nodes of lymphatic vessels into the venous blood [51,52,53,54].

High-speed video lymphoscintigraphic measurements showed the velocity of lymph flow in the ~2-9 mm/s range, which implies that the small portion (about 3 %) of Comirnaty nanoparticles that does not reside in the deltoid muscle and the draining lymph nodes, reaches the blood within about 1 hour [55,56]. As additional variables that contribute to the individual variation of vaccine pharmacokinetics, the pumping activity of lymph microvessels are delicately controlled by nitric oxide and prostaglandins released from the endothelium [51]. Figure 3 details the anatomical roadmap of mRNA–LNPs transport from the deltoid muscle to the blood.

A third potential route for LNP entry into the bloodstream involves concentration gradient-driven transcapillary transport from the interstitial space into capillaries, particularly in regions where vascular permeability is elevated due to the proinflammatory effects of the vaccine. This process can be considered a reversal of the enhanced permeation and retention (EPR) effect, which is exploited for passive targeting of liposomes in cancer chemotherapy [57]. As for the extent and kinetics of systemic biodistribution of mRNA-LNPs, Pfizer Australia's preclinical study reported that 2.8% of a radioactive lipid marker remained in the plasma of rats 15 minutes after intramuscular injection of Comirnaty-equivalent LNPs, with plasma levels peaking between 1 and 4 hours [58].

7. Quantitative Aspects of Vaccine Nanoparticle-Endothelium Engagement

The vascular endothelium constitutes the first biological barrier to the systemic distribution of mRNA–LNPs, prompting the key question of stoichiometry: how many mRNA-LNPs actually reach and interact with the apical surface of each endothelial cell? A calculation at 15 minutes post-injection suggests that approximately 500 to 7,000 mRNA–LNP may potentially interact with each endothelial cell in the body. This implies that a single vaccine dose delivers mRNA–LNPs in substantial excess relative to the number of endothelial cells. The assumptions underlying this estimate are as follows: (i) each 30 µg dose of mRNA in a Comirnaty injection contains approximately 2 × 10⁻⁸ moles of mRNA, based on a molecular weight of ~1.4 MDa, corresponding to roughly 1.2 × 10¹³ mRNA molecules; (ii) approximately 3% of the injected mRNA–LNPs reach the bloodstream within 15 minutes [58]; (iii) each LNP carries 1–2 mRNA molecules [59,60,61,62,63]; (iv) the total endothelial surface area in the human body is 1–7 m², equivalent to 1–7 × 10¹² µm²; and (v) the apical surface area of a typical endothelial cell is 300–600 µm² [64,65].

It must be emphasized, however, that this is a snapshot calculation; the number of mRNA–LNPs available for interaction with endothelial cells rapidly declines due to uptake by various organs and depends critically on the rate of entry into the circulation. The key implication of these metrics is that vaccine-induced endothelial transfection, activation, and consequent inflammation could potentially affect any segment of the vascular endothelium. Hence, the small amount of vaccine inoculum does not preclude the possibility of off-target distribution of vaccine nanoparticles.

8. The Pathophysiology and Diagnosis of Vaccine-Induced Vasculitis

Table 3 shows the types and symptoms of vasculitis classified according to the size of the blood vessels affected. Based on the proportional relationship between LNP uptake and capillary surface area, most vasculitis symptoms are associated with inflammation of small vessels, which collectively provide the largest endothelial interface. Nevertheless, the inflammatory diseases involving tissue compartments enriched in medium- and large-sized blood vessels have also been documented to display distinct clinical features [66,67,68,69] (Table 3).

The diagnosis of vasculitis-related illnesses on organ level remains challenging. The nonspecific symptoms, such as fever, fatigue, rash, may not pin down the organ affected just as the inflammatory markers (e.g., CRP) and autoimmune indicators (e.g., ANCA, ANA) in blood or cerebrospinal fluid. Imaging studies, including conventional or digital subtraction angiography, MRI with contrast and tissue biopsies support the diagnosis, but a thorough differential diagnosis is essential to exclude infections, malignancies, and thrombotic disorders or ischemic or hemorrhagic [70]. Advanced techniques like vessel wall imaging (VWI) and magnetic resonance angiography (MRA) may reveal vessel abnormalities [71].

Focusing on the role of the spike protein in post-vaccination cerebral arteritis, a recent study demonstrated its prolonged presence in cerebral arteries, accompanied by inflammatory cell infiltration [72]. This chronic cerebral inflammation may disrupt local circulation and oxygen delivery, potentially underlying symptoms such as fatigue, memory loss, dementia, Alzheimer acceleration and other cognitive or psychological complaints following vaccination [28,73]. Supporting this, we observed impaired cerebrovascular regulation in individuals with post-COVID condition (whether from infection or vaccination), assessed using transcranial Doppler ultrasound, a non-invasive method that measures blood flow velocity in major cerebral arteries.

As shown in Figure 4, the attenuated reactive hyperemic response reflects diminished vasodilatory capacity of cerebral resistance vessels in the brain and was associated with persistent cognitive and mental dysfunction even after ~2 years. Notably, this impairment was alleviated by regular physical activity [28].

9. Non-Target Organ Uptake of mRNA–LNPs

Beside the classic endocytic uptake of mRNA-LNPs by phagocytic cells in the body, a main mechanism is transfection via fusion. This function of LNPs has been known since the discovery in the late 80-s that the ionizable, positively charged lipids can tightly bind the negatively charged nucleic acids and carry them into the cytoplasm of cells without loss of gene function [61,74,75,76,77,78,79,80,81]. Since then, many different tissues and cells have been shown to be “viable targets” for gene therapies using LNPs [82], providing rationale for using the LNPs for the delivery of spike protein mRNA to immune cells in the vicinity of the injection; the widely claimed mode of action of mRNA vaccines.

In the background of the selection of the ionizable positively charged aminolipids (ALC-0315 in Comirnaty) as main LNP component in mRNA vaccines, the concept was based, among others, on its nucleic acid-binding capability with fusogenic activity resulting in unique competency for genetic modification of cells. This selection was in keeping with the clinical success of liver-targeted patisiran (Onpattro), the first FDA-approved gene therapy against amyloidosis [79].

The assumption that Comirnaty delivers mRNA exclusively to antigen-presenting cells (APCs) near to the injection site may have overlooked a 2015 study by Pardi et al, which showed delivery by LNPs and translation of firefly luciferase-mRNA in cells at the injection site and its draining lymph nodes, as well as in the liver, lung and other organs within 5 hours [83]. In Pfizer Australia's preclinical rat study, beyond the injection site and sentinel lymph nodes, the LNPs were primarily distributed to the liver, adrenal glands, spleen, and ovaries over a 48-hour period, and low-level (<2%) radioactive signals were also detected in 12 additional organs [58].

In further large-animal studies, Ferraresso et al. [82] demonstrated that LNPs transfected nearly all major organs in pigs. More recently, Dézsi et al. [84] reported the presence of both spike protein mRNA and the translated spike protein in multiple organs as early as 6 hours following intravenous injection of Comirnaty. Notably, the uptake of spike protein mRNA showed a close temporal correlation with the transcriptional upregulation of key proinflammatory cytokine genes, including IL-6, TNF-α, and IL-1. These findings align with the known proinflammatory properties of both the mRNA and the spike protein, although it remains unclear whether one plays a dominant role in cytokine induction or whether they act synergistically.

The internalization of LNPs by different cells may occur through multiple pathways, including clathrin- or caveolae-mediated endocytosis, micropinocytosis -particularly in inflamed or activated endothelium- and/or membrane fusion, a hallmark of LNPs containing ionizable aminolipids. As for ECs, experimental evidence for effective LNP delivery of functional mRNA into these cells across various organs is well documented [85,86,87,88,89,90,91,92]. However, regardless of the entry mechanism of mRNA-LNPs into the ECs, the internalized mRNA and de novo synthesized spike protein trigger the activation of ECs via a variety of ways, direct and indirect, independent and cooperative, involving innate and adaptive immune responses. These are itemized below.

10. Adverse Impacts of mRNA-LNPs and the Spike Protein on Endothelial Cells

The spike protein functions as a pluripotent toxin [93,94,95,96,97], causing oxidative damage of the mitochondria [98,99,100], induction of proinflammatory cytokines [101,102] and other self-destructive effects in the cells that produce them.

Further immune-mediated damage to the mRNA-LNP transfected EC is related to the diversification of the intracellular antigen processing of free ribosome-derived spike protein [9,103]. Notably, after formation on free ribosomes, which exceeds the number of endoplasmic reticulum (ER)-bound ribosomes in most cells [104], a part of the intact spike protein molecules in transfected cells undergoes proteasome digestion, which yields antigenic peptides to be transported to MHC Class I molecules on the endothelial cell surface [103]. This process is known as cross-presentation [105], since the spike protein degradation peptides are supposed to be presented on MHC-II molecules. [105],. In previously infected or immunized people, MHC Class I-presented peptides are recognized by specific cytotoxic T cells (Tc, CTLs) which attack the EC cells (Figure 5). This mechanism plays a major role in the widespread autoimmune phenomena observed after mRNA-type vaccination and may help explain why booster injections can elicit more pronounced AEs than primary immunization.

In addition to proteasomal degradation, an alternative trafficking route for the translated spike protein directs it to the cell membrane, i.e., the same destination it naturally follows in SARS-CoV-2-infected cells. As a result, the luminal surface of ECs, along with the outer membrane of other transfected cells, becomes “crowned” with the spike protein, mimicking a true viral infection. The immune system recognizes these “pseudo-infected” cells and targets them through at least two antibody-dependent elimination mechanisms: complement (C) activation (Figure 6) and antibody-dependent cellular cytotoxicity (ADCC) (Figure 7).

Regarding C activation, Comirnaty is a strong initiator of this process [106,107,108], a relatively ignored contributor to the AEs of mRNA vaccines [63]. It came to the focus of attention in this subject because of its role in the acute anaphylactic reactions whose incidence had significantly increased after immunisation with mRNA vaccines [109]. These investigations [63,106,107,108,109] led to the concept that C activation-related pseudoallergy (CARPA) represents a significant contributor to vaccine-induced anaphylaxis [63]. However, while CARPA is due to fluid phase activation of the proteolytic cascade, the C-mediated cytotoxicity proceeds on cell surfaces, including the ECs. C activation on cell surfaces that express the spike protein and or bind PEGylated LNP can also occur via both the classical and the alternative the pathways [107]. The former process may be due to anti-PEG antibody binding to EC-adhered mRNA-LNPs, which express PEG on their surface (Figure 6A). The spike protein is moving from free ribosomes to the cell surface, triggering also classical pathway C activation through the binding of anti-spike protein antibodies Figure 6B). In addition, fluid phase and cell surface spike protein can induce lectin pathway activation [96,110] (Figure 6C) and the damaged cell membrane can bind C3b directly, the core mechanism of alternative pathway activation (Figure 6D). Each of these activations can be perpetuated via the alternative pathway amplification loop [111,112] fed by activated EC-produced C3, properdin and factor B.

The other antibody-mediated EC cell damage is called antibody-dependent cellular cytotoxicity (ADCC) whereupon effector cells of the immune system recognize and eliminate target cells that have been opsonized by specific antibodies. The cells involved in this action include natural killer (NK) cells, macrophages and neutrophils. The specific antibodies, usually IgG1 or IgG3, bind to antigens expressed on the surface of a target cell and these antibodies are recognized by the Fcγ receptors (especially FcγRIIIa/CD16) on the above cells (Figure 7). Binding of FcγR triggers degranulation of these effector cells, just as the binding of CTL to MHC Class I molecules triggering the release of cytolytic mediators, namely perforins and granzymes. The latter proteins, especially granzyme B, are serine proteases that activate caspases (e.g., caspase-3) that cause DNA fragmentation and apoptosis. Granzyme A induces caspase-independent cell death (via reactive oxygen species, mitochondrial damage). Both NK cells and CTLs use receptor-ligand interactions leading to cell death, such as the binding of Fas Ligand (FasL, CD95L) to Fas receptors (CD95) on target cells, activating caspase-8 and causing apoptosis [113,114]. The TNF-related apoptosis-inducing ligand (TRAIL) is also involved in the apoptotic attack of Tc and/or NK on the ECs [115]. Among the cytokines produced, IFN-γ enhances macrophage activity and antigen presentation [116].

11. Causes and Features of Endothelitis

Figure 8 illustrates the process whereupon the encounter with vaccine nanoparticles entails the activation of ECs. In this process, the glycocalyx derangement enables the deposition of neutrophils, monocytes and macrophages, with the release of oxygen radicals, PLA2, cytokines, leukotrienes, C proteins and other inflammatory mediators. The pathophysiological manifestations of EC activation and damages include increased vascular permeability, opening the gap junctions, release of PLA2, leukotrienes, cytokines and reactive oxygen species, deposition of neutrophils, monocytes and macrophages. The opening of the gap junctions upon vasodilatation (which increases intravascular pressure) is associated with oedema and pain. The increased production of thromboxane A2 and reduced production of prostaglandins and nitric oxide create a pro-coagulant environment promoting thrombogenesis, which is frequently observed after mRNA-type vaccinations. Moreover, damaged ECs release DAMPs, which further activate the immune system and contribute to a cycle of self-perpetuating chronic condition. Endothelial microparticles (exosomes) and anti-endothelial cell antibodies have been identified as diagnostic or prognostic biomarkers of endothelitis.

12. Outlook

Since the introduction and widespread deployment of mRNA-based COVID-19 vaccines, their safety profile has become a focal point of both public discourse and scientific investigation. This cutting-edge technology has raised several unresolved questions, particularly regarding the unusually broad spectrum and relatively high incidence of AEs associated with its use [1]. According to an evolving paradigm shift, beyond their intended antiviral efficacy, COVID-19 mRNA vaccines may cause collateral tissue damage due to inherent design limitations, as elaborated in Section 2 of this review on mRNA–LNP-induced inflammatory complications. In essence, the unique properties of the spike protein antigen and its delivery vehicle, the mRNA-LNP lipoplex, can provoke immune responses resembling those of active infection. Thus, the vaccine nanoparticles act as non-replicating, functionally simplified mimics of SARS-CoV-2 infection and, as such, exhibit features akin to live attenuated viruses with potential for reverted pathogenicity [1,9].

Although -fortunately-, the AEs affect only a small subset of vaccine recipients, safety concerns have begun to influence vaccination policies. Most recently, the CDC and FDA ceased recommending COVID-19 mRNA vaccination for children and pregnant women, restricting its use primarily to high-risk populations [117,118]. At this time, the attention is being centered on the accumulating evidence of AEs and their public health implications, while the biological roots of the health problem is far from being understood. Among scientific investigations into vaccine-related AEs, there is growing recognition that a common pathogenic denominator is vascular inflammation, specifically, endothelitis. This review aims to advance understanding of the problem by focusing on tissue-specific features of vaccine-transfected microcirculation, elucidating the mechanisms of mRNA-LNP intravasation and cellular uptake, and examining the self-injurious consequences of spike protein expression. While acknowledging the public health achievements and benefits of mRNA-LNP-based vaccines, the insights gained from studying their AEs will be instrumental in improving the safety of future vaccines and therapeutics using this platform. A key unresolved question for future research is: what protective mechanisms shield the vast majority of vaccine recipients from these complications?